Phage derived antimicrobial activities

ABSTRACT

The present invention provides methods and compositions to reduce growth of microbial colonies, including infections, and includes therapeutic compositions, methods for treatment of infections, and methods for identifying additional such compositions.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/797,885, filed May 5, 2006 and U.S. Provisional Application No.60/909,340, filed Mar. 30, 2007; both of which are herein incorporatedby reference for all purposes.

FIELD OF INVENTION

The present invention provides methods and compositions to reduce growthof microbial colonies, including infections, and includes therapeuticcompositions, methods for treatment of infections, and methods foridentifying additional such compositions.

BACKGROUND OF THE INVENTION

Bacteria are ubiquitous, and are found in virtually all habitableenvironments. They are common and diverse ecologically, and find unusualand common niches for survival. They are present throughout theenvironment, and are present in soil, dust, water, and on virtually allsurfaces. Many are normal and beneficial strains, which provide asynergistic relationship with hosts. Others are not so beneficial, orcause problems along with benefits.

Pathogenic bacteria can cause infectious diseases in humans, in otheranimals, and also in plants. Some bacteria can only make particularhosts ill; others cause trouble in a number of hosts, depending on thehost specificity of the bacteria. Diseases caused by bacteria are almostas diverse as the bacteria themselves and include food poisoning, toothdecay, anthrax, general infectious diseases, and even certain forms ofcancer. These are typically the subject of the field of clinicalmicrobiology.

Bacteria are killed in nature by bacteria-specific viruses, e.g.,bacteriophage, or phage. Many phages found in nature belong to the groupCaudovirales, or “tailed” phages. These viruses invariably have a singledouble-stranded DNA genome packaged into a proteinaceous capsid. Thephage consists of three fundamental structures: the head; which ingeneral has icosahedral symmetry, a tail structure emanating from onevertex of the icosahedral head, and 4-6 tail fibers attached to somepart of the tail. It should be noted that the Order Caudoviralescontainsthree general morphotypes: Podoviridae (podophage), Myoviridae(myophage), and Siphoviridae (siphophage). Strictly speaking, thepodophage do not have a morphogenetically separate “tail”; that is, thetail-like structure is actually assembled as part of head or capsidassembly. In the myophage and siphophage, there are separatemorphogenesis pathways for heads, tails and tail fibers; all three areeventually joined together to form complete infectious virions. Inpodophage, there is a head pathway and a tail fiber pathway. From afunctional perspective, however, the tail like structure of podophageserves the same function as the genuine tails of the other twomorphotypes.

Phages kill cells by infecting, replicating, and then lysing the hostcell, releasing multiple progeny virions in the process. Certainphage-derived elements are also capable of killing cells. For example,many Pseudomonas strains produce pyocins, proteinaceous components thatkill other Pseudomonas strains. In general, the term “bacteriocin” isused to describe compounds produced by bacteria that kill otherbacteria; bacteriocins of a wide-variety of chemical structure, fromsmall molecules to polypeptides, are known. However, many of the pyocinswere found to be “headless tails”, i.e., phage tails produced withoutheads or DNA. These tail-like bacteriocins kill bacteria by adsorbing tothem and causing a fatal lesion in the cell envelope, although, lackingDNA, there is no replication or host lysis. Since the original discoveryof the pyocins in Pseudomonas, similar tail-like bacteriocins have beenidentified in a wide variety of other bacteria, including bothGram-negative and Gram-positive species. See, e.g., Nakayama, et al.(2000) Mol. Microbial. 38:213-31; Traub, et al. (1996) Zentralbl.Bakteriol. 284:124-35; Ito, et al. (1986) J. Virol. 59:103-111; Rocourt(1986) Zentralbl. Bakteriol. Mikrobiol. Hyg. 261:12-28; Shinomiya (1984)J. Virol. 49:310-14; Ishii, et al. (1965) J. Mol. Biol. 13:428-431; Dawand Falkiner (1996) Micron. 27:467-479; Strauch, et al. (2001) Appl.Environ. Microbiol. 67:5635-5642; and Abdelhamid, et al. (2002) Appl.Environ. Microbiol. 68:5704-5710. In addition, other bactericidalelements derived from phage have been described. For example,Caudovirales encode an endolysin as part of the host cell lysisfunctions. These enzymes degrade the host cell wall from within, leadingto lysis and release of the progeny virions. Phage endolysins addedexogenously to cultures or suspensions of bacteria have been shown to becapable of lysing and killing a number of Gram-positive bacteria. See,e.g., Fischetti, et al. (2005) US Pat App 20050208038 describing use ofphage endolysins to kill bacteria and Takac and Blasi (2005) Antimicrob.Agents and Chemother. 49:2934-2940.

Certain bacteria are normally innocuous, but become pathogenic uponpresentation of the appropriate opportunity, or become problematic uponintroduction to an abnormal site or situation. Persons lacking effectiveimmune systems are most vulnerable, and certain bacteria use susceptibleweak hosts to provide a temporary environment to proliferate anddisperse throughout the population.

Statistically, infectious diseases are a major medical problem. See,e.g., Watstein and Jovanovic (2003) Statistical Handbook on InfectiousDiseases Greenwood, ISBN: 1573563757. In the U.S., some 40-70K deathsresult from bloodstream nosocomial (hospital derived) infections eachyear.

Antibiotics have revolutionized clinical medicine over the last halfcentury. Since the original discovery of antibiotic phenomenon, themechanism of action and development of this class of remarkabletherapeutic entities has made enormous progress. See, e.g., Therrien andLevesque (2000) FEMS Microbiol Rev. 24:251-62; Durgess (1999) Chest115(3 Suppl):19S-23S; Medeiros (1997) Clin. Infect. Dis. 24(Suppl1):S19-45; Jones (1996) Am. J. Med. 100(6A):3S-12S; Ford and Hait (1993)Cytotechnology 12:171-212; and Liu (1992) Compr Ther. 18:35-42.Antibiotics had about $32B worldwide sales in 2002.

The widespread appearance of antibiotic-resistant bacteria hasemphasized the vulnerability of current antimicrobial treatments tobacterial adaptation. See, e.g., Walsh (1992) Antibiotics: Actions,Origins, Resistance Amer. Soc. Microbiol., ISBN: 1555812546; Cunha(1992) Antibiotic Essentials Physicians Press, ISBN: 1890114413; Amyes(2003) Magic Bullets, Lost Horizons: The Rise and Fall of AntibioticsTaylor & Francis, ISBN: 0415272033; Axelsen (2001) Essentials ofAntimicrobial Pharmacology: A Guide to Fundamentals for Practice HumanaPress, ISBN: 0896038424; and Mainous and Pomeroy (eds. 2001) Managementof Antimicrobials in Infectious Diseases: Impact of AntibioticResistance Humana Press, ISBN: 0896038211. However, many classicalantibiotics require rapid replication or growth of the target bacteriato be effective.

Thus, improved methods for decreasing target bacterial growth orsurvival or limiting bacterial pathogenicity will find great utility.This utility may be applicable to environmental, local, topical, orparticularly in vivo colonization. The present invention addresses theseand other significant problems.

BRIEF SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery thatphage-encoded cell wall degrading activities, e.g., murein-degrading(commonly designated “muralytic”) enzymes, which are typically the coreof the phage lysis functions to exit the host cell, are also found asstructural components of the phage virion and assist entry of the phageinto a host cell. These activities, designated here as TAMEs(tail-associated murein-degrading enzymes), have intrinsic bactericidalactivity, irrespective of the phage replicative pathway. Each phageparticle of all three morphotypes is thought to have a TAME associatedwith the tail structure or, in the case of the podophage, associated asa minor component of the head or capsid. It is thought that localdegradation of the cell wall facilitates the DNA injection process. Theinvention describes a particular phage TAME, ORF56, the product of orf56of the staphylococcal myovirus K. In particular, purified ORF56,heretofore not recognized as a “lytic” agent, is found to havebactericidal activity. Moreover, bactericidal polypeptides derived fromORF56 by truncation have been identified. Bactericidal activity can bescreened for from similar or related sources, e.g., sources of similarstructures and domains from various evolutionarily diverse sources, tofind additional bactericidal activities which possess advantageousproperties. Such sources may also be starting points for mutagenesis andscreening for additional advantageous properties, e.g., stability,bactericidal efficiency, size, substrate specificity, and such. Mostimportantly, robust bactericidal activity, significantly (e.g., ordersof magnitude, or multiple factors) more efficient than found for thepurified TAME ORF56 or its truncation derivatives, is found for achimera consisting of the murein-degrading catalytic domain of ORF56 andthe non-catalytic cell-wall binding domain (CBD) of the lyticStaphylococcal bacteriocin, lysostaphin. These TAME-CBD chimeras aremuch more efficient in terms of bactericidal activity than the purifiedTAME. Moreover, the TAME-CBD chimeric protein is shown to persist in anefficacious state (e.g. retains enzymatic stability), in terms ofbactericidal activities, in a number of useful formulation mixtures.Purified proteins based thereon, and nucleic acid sequences encodingsuch are provided, along with antibodies thereto. Methods for using saidcompositions are provided, including methods to reduce the growth orpresence of the target bacteria.

The present invention provides a method of killing a bacteriumsusceptible to a cell wall degrading activity, said method comprisingintroducing to the environment of said bacterium a composition selectedfrom: a) a purified TAME component of a phage tail or a tail-likebacteriocin; b) a cell wall degrading portion of the phage K ORF56 TAMEor the presumptive TAME of phage phi11, ORF49; c) a substantially purepolypeptide comprising a cell wall degrading polypeptide of phage KORF56 or phage phi11 ORF49; or d) a pharmaceutical compositionconsisting essentially of the TAME homologs, or fragment thereof, fromother phages or tail-like bacteriocin. Examples of sources include agroup consisting of: YP_(—)238566 (ORF007 (Staphylococcus phage Twort)),YP_(—)406405 (gp29 (Listeria bacteriophage P100)), NP_(—)765044(secretory antigen SsaA-like protein (Staphylococcus epidermidis ATCC12228)), YP_(—)164769 (orf134 (Lactobacillus plantarum bacteriophageLP65)), YP_(—)492702 (transfer complex protein TraG (Staphylococcusaureus subsp. aureus USA300)), AAA71958 (putative (Staphylococcusaureus)), NP_(—)765786 (N-acetylmuramoyl-L-alanine amidase(Staphylococcus epidermidis ATCC 12228)), YP_(—)189676 (secretoryantigen precursor SsaA-related protein (Staphylococcus epidermidisRP62A)), YP_(—)189814 (N-acetylmuramoyl-L-alanine amidase(Staphylococcus epidermidis RP62A)), and other designated sourcesfurther described below. Another source is the phage phi11 ORF49, aputative cell wall hydrolase (NP_(—)803302; GeneID:1258067).

The invention further provides methods, as described, wherein: thebacterium belongs to genus Staphylococcus; and specifically is S.aureus, S. epidermidis and other staphylococci of clinical significance;the environment is in vivo or on a mucosal or other organ surface or ona medical device or implant; the introducing is topical, systemic,parenteral, or by inhalation; another antimicrobial treatment is used,including an antibiotic or phage-derived product; or said bactericidalactivity has a broad target specificity across multiple bacterialstrains and/or across multiple bacterial species.

Various methods are provided for screening for a phage-derivedbactericidal activity on a target bacterium, said method comprising:fragmenting a source phage into separable structural fragments;determining which fragments retain binding affinity for said targetbacterium; and testing said fragments for bactericidal activity; therebyidentifying structures possessing said bactericidal activity. This alsoincludes embodiments wherein the data from the method is communicatedinto a US jurisdiction. In certain embodiments, the target bacterium isa Gram-positive bacterium or the bactericidal activity is a muralyticactivity.

More methods are provided, including one for generating variantbactericidal activities, the method comprising mutagenizing a geneencoding a polypeptide characterized as exhibiting cell wall degradingactivity; and screening for variants with modified bacteriocidalactivity. Communicating the data from such a method would also beencompassed. Other methods include that described, but evaluating formodified bactericidal activity, e.g., different substrate turnovernumber; or a change in sensitivity of enzymatic properties to reactionconditions, including temperature, salt, pH, hydration, or the like.

Treatment methods are provided, including one of treating a bacterialinfection in an animal, the method comprising administering to saidanimal one or more bactericidal polypeptides, wherein at least two ofsaid polypeptides are derived from different cell wall degrading genes;the bactericidal polypeptides have broad target bactericidal activity;the bactericidal proteins are “lytic” when applied to the exterior ofthe cell; or the bactericidal activity is murein-degrading, ormuralytic, which includes proteins with murein glycosidase (includingglucosaminidase and muraminidase), transglycosylase, lysozyme, amidaseor endopeptidase activities.

The present invention provides an ORF56 or ORF49 polypeptide that hasbactericidal activity against a target bacterium and that, at a minimumincludes an amino acid sequence with at least 80%, 90% or 95% identityto amino acid residues 620-808 of SEQ ID NO: 1 or residues 481-618 ofSEQ ID NO: 3. In one embodiment, the ORF56 protein includes the exactsequence of amino acid residues 620-808 of SEQ ID NO: 1 or the ORF49protein includes the exact sequence of amino acid residues 481-618 ofSEQ ID NO: 3. In a further embodiment, the invention provides acomposition that consists essentially of an ORF56 polypeptide that hasbactericidal activity against a target bacterium and that, at a minimum,includes an amino acid sequence with at least 80%, 90%, or 95% identityto amino acid residues 620-808 of SEQ ID NO: 1 or residues 481-618 ofSEQ ID NO: 3.

In one embodiment, the invention provides a composition, e.g., apharmaceutical composition, a diagnostic reagent, or a bactericidalcomposition, that includes an ORF56 or ORF49 polypeptide that hasbactericidal activity against a target bacterium and that includes anamino acid sequence with at least 80%, 90%, or 95% identity to, at aminimum, amino acid residues 620-808 of SEQ ID NO: 1 or residues 481-618of SEQ ID NO: 3. The composition can include at least one other proteinwith bactericidal activity, e.g., a p16 protein from phage p68 or aPal-type “lytic enzyme”. The composition can also include otheringredients with bacteristatic or bactericidal activity, e.g., anantibiotic.

The disclosed ORF56 or ORF49 polypeptides can be used to prevent growthof a target bacterium that is a Staphylococcus species, and inparticular a methicillin-resistant Staphylococcus species. In anotherembodiment, the target bacterium is a slowly replicating bacterialspecies, e.g., a bacterium that has a doubling time between one andseventy-two hours, or more, e.g., about 2, 4, 8, 12, 20, 30, 40, or 50hours.

The disclosed ORF56 and ORF49 polypeptides or a composition thatincludes an ORF56 or ORF49 polypeptide can be used to, e.g.,enzymatically degrade a bacterial cell wall.

In another aspect the invention provides a method of treating abacterial infection in a subject by administering an ORF56 or ORF49polypeptide or a composition that includes an ORF56 or ORF49 polypeptideto the subject. The subject can be, e.g., a mammal, a primate, a human,a farm animal, a companion animal, a human, a poultry species, a cow, ahorse, a goat, a cat, a sheep, a rodent, a dog, a pig, a chicken, aduck, a quail, or a goose. Show animals, e.g., elephants, lions, tigers,zebras, whales, dolphins, and bears can also be treated using thecompositions of the present invention.

In various embodiments, the subject is a cow and the bacterial infectionis bovine mastitis; the subject is a human and the bacterial infectionis caused by a methicillin-resistant Staphylococcus species; or thesubject is a poultry species and the bacterial infection is on the skinor feathers.

In another aspect the invention provides a method detecting a bacteriumor identifying a disease causing bacterium by contacting the bacteriumwith an ORF56 or ORF49 polypeptide and detecting binding of the ORF56 orORF49 polypeptide to the bacterium. In a preferred embodiment the ORF56or ORF49 polypeptide is detectably labeled.

In one aspect the invention provides a method of disinfecting a surface,by contacting the surface with an ORF56 or ORF49 polypeptide or acomposition that includes an ORF56 or ORF49 polypeptide. Thedisinfection method can be used to reduce or eliminate all bacteria onthe surface or a plurality or a particular bacterial species or strain,e.g., a Staphylococcus species.

In one aspect the invention provides a substantially pure or isolatedpolypeptide characterized by at least one of the following properties:comprising at least about 85% identity over a segment of at least 17amino acids to residues 1-808, 297-808, 363-808, 603-808, 620-808, or691-805 of ORF56; comprising at least about 90% identity over a segmentof at least 24 amino acids to residues 691-805 of ORF56; or comprising aplurality of distinct segments of a least 85% identity to ORF56, whichsegments do not overlap. Some additional properties include, e.g.,distinct additional segments of at least about 75% identity over atleast 17 amino acids to residues 691-805 of ORF56; distinct additionalsegments of at least 17 amino acids exhibiting at least about 65%identity over ORF56; at least 30% cell wall degrading activity of fulllength or native ORF56; a muralytic activity on a Staphylococcusbacterial strain at least about 50% of ORF56; another functionalpolypeptide sequence or domain, e.g., a signal sequence; or a detectablelabel; comprises at least residues 690 to 769 of ORF56; is a full lengthORF56; corresponds to 1-808, 297-808, 363-808, Met-603-808, or 620-808of ORF56; comprises a CHAP domain; is substantially free of other phageproteins; is substantially free of other proteinaceous materials; iscombined with another antimicrobial agent, including an antibiotic; isadmixed with a pharmaceutical excipient; is in a buffered or sterilecomposition; exhibits a bacterial cell wall degrading activity selectedfrom muralytic, glucosamidase, amidase, or endopeptidase activity;exhibits bactericidal activity on multiple Gram-positive bacteriastrains; exhibits bactericidal activity on a Staphylococcus bacteriastrain; or exhibits bactericidal activity on one or more strainsdescribed as S. aureus, S. epidermidis, S. lentis, S. simulans, and S.carnosus.

In one aspect the invention provides an expression vector that expressesan isolated or recombinant nucleic acid that encodes an ORF56 or ORF49polypeptide or a truncation of an ORF56 polypeptide disclosed herein.The invention also includes host cells that contain the ORF56 expressionvector. A host cell can be, e.g., a eukaryote or prokaryote cell that isused to produce an ORF56 polypeptide or nucleic acid.

In one aspect the invention provides a substantially pure or isolatedORF56 polypeptide that has an antigen binding site of an antibody thatbinds selectively to a cell wall component. This ORF56 polypeptide canbe, e.g., attached to a detectable label or provided as part of a kitwith instructions that is used to evaluate the presence of targetbacteria.

In one aspect the invention provides a method of enzymatically degradingthe cell wall of a target bacterium, by exposing said cell wall to anORF56 or ORF49 polypeptide. This method step can, e.g., be incorporatedinto a diagnostic to determine bacterial sensitivity; resulting in atleast about a 5-fold decrease in sensitive bacterial population on awork or furniture surface; introduce the ORF56 or ORF49 polypeptide intoan animal and results in at least a 5-fold decrease in sensitivebacterial population in a selected location in or on said animal;administer said polypeptide to an animal surface or compartment; be ameans to generate dead or replication incompetent bacteria that can beinoculated into an individual; or be used to treat a skin, mucosal,urinary tract, respiratory tract, nasal cavity, gastrointestinal tract,or other bacterial infection. In other embodiments the decrease in asensitive bacterial population is, e.g., a 2-fold, 3-fold, 4-fold,7-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-folddecrease, or even more.

In one aspect the invention provides a recombinant truncated ORF56protein of SEQ ID NO: 1, wherein from 1 to 620 amino acids are truncatedfrom the amino terminus of the ORF56 protein or wherein from 1 to 3amino acids are truncated from the carboxy terminus of the ORF56protein, and wherein the ORF56 protein has bacterial cell wall degradingactivity. The remaining ORF56 protein can have about 80%, 90%, or 95%identity to the corresponding amino acid sequence in SEQ ID NO: 1.

The present invention provides a substantially pure or recombinantpolypeptide exhibiting Staphylococcus strain murein degrading biologicalactivity, the polypeptide comprising a tail associated murein-degradingenzyme (TAME) segment of a S. aureus infecting phage; and a heterologousS. aureus cell wall binding domain.

In certain preferred embodiments, the polypeptide has a protein backbonemolecular weight of less than about 400 kDa, about 250 kDa; or about 100kDa; or the polypeptide exhibits a peptidase, amidase, or hydrolaseactivity on a S. aureus murein; or the polypeptide is from the tail of aCaudovirales phage, e.g., a myoviridae, podoviridae, or siphoviridaephage. In other embodiments, the murein-degrading enzyme segment is fromphage K ORF56, phage phi11 ORF49, or a phage derived from an MRSA. Invarious other embodiments, the cell wall binding domain is from aStaphylococcus bacterial protein, e.g., a Staph lysostaphin or a phagetail protein; or comprises: a bacterial SH3 segment; sequence fromORF56, S. simulans lysostaphin, or Phage L54a amidase; or any cell wallbinding domain construct that increases murein-degrading activity of thepolypeptide at least by 30 fold, as compared to a comparable polypeptidelacking function of the binding domain.

In one embodiment, the polypeptide comprises SEQ ID NO: 4.

Pharmaceutical compositions are also provided, e.g., where thepolypeptide is in a cream or gel, or is in a single dose container,e.g., containing at least 10 nanogram of polypeptide. Such compositionsmay be in a controlled release formulation; applied to an implant,catheter, or medical device; or be in a sterile or buffered formulation.

In other preferred embodiments, the composition works on aStaphylococcus strain that is found in a nasal compartment; or thatcauses mastitis or infects burn or puncture wounds; or that ismethicillin resistant or that is Vancomycin resistant. In anotherpreferred embodiment, dressings, gauzes or the like used to cover woundsare impregnated with a TAME polypeptide or a chimeric protein comprisinga TAME polypeptide to minimize the likelihood of bacterial infection. Ina further embodiment, the wound is a puncture wound or a burn. In yetanother embodiment, the individual with the wound has compromised immunesystem, e.g., resulting from HIV infection, organ transplantation andrelated treatments, stem cell or bone marrow transplantation, orchemotherapy. The TAME polypeptides and chimeric proteins comprising aTAME polypeptide can also be used to treat organs or blood productsbefore transplantation into a recipient.

The invention further provides methods of treating a bacterial culture,the method comprising contacting said culture with a described chimericpolypeptide. Typically, the contacting decreases rate of growth of saidculture by at least about 5 fold; or another antimicrobial therapy isalso used; or the method uses a cocktail of polypeptides which targetdifferent strains of bacteria. Preferably, the treating decreases rateof growth of sensitive target bacteria by at least about 30%; thepolypeptide is administered at a stoichiometry of at least about tenpolypeptides for each target bacterium, or at least about 500 ng/ml; orthe contacting of administering is continued for less than about 7 days.Alternatively, the culture comprises a Staphylococcal strain, aGram-positive bacterium, or is an infection, or the culture may comprisemammalian cells or tissue.

The invention further provides a nucleic acid encoding the polypeptides,though the polypeptides may be generated by synthetic protein methods.And a cell comprising the nucleic acid is provided.

The invention further provides equivalent or related polypeptidesderived from fusions of the TAME polypeptide sequence, or a segment ofthe TAME sequence containing a muralytic domain, to a segment of anotherpolypeptide constituting a cell wall binding domain (CBD). Thesechimeric constructs will be designated as TAME-CBDs. The e.g., asubstantially pure or recombinant polypeptide exhibiting Gram-positivestrain murein-degrading biological activity, where the polypeptidecomprises a modified, e.g., mutagenized, sequence of a Tail AssociatedMurein-degrading Enzyme (TAME) segment of a Gram-positive infectingphage; and/or a modified, e.g., mutagenized, heterologous Gram-positivecell wall binding domain. In these cases, “mutagenized” is primarily aform of the TAME in which regions of the complete TAME protein have beendeleted, with the effect of increasing bactericidal or enzymaticactivity, protein solubility, and/or protein stability, compared to thefull-length TAME.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a table of TAME conserved domains identified in phagesthat infect Staphylococcus bacteria. Muralytic domains (MD) wereidentified and are referred to as TAME CD in the able.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present study identified a heretofore unidentified entity, the phageK ORF56, which has now been shown to exhibit bactericidal activity. Itis a component of certain myoviridae phage as a structural component ofthe virion with muralytic activity. The catalytic site was localized inthe C-terminal part of the protein, and exhibits a Cysteine-Histidinedependent Aminohydrolase/Peptidase (CHAP) domain. See, e.g., Rigden, etal. (2003) Trends Biochem. Sci. 28:230-234. While the CHAP domain isfound at the N-terminal regions of various genes, in a few genes theCHAP domain is found at the C-proximal segments of coding regions. Partof the invention is the understanding of a relationship betweencharacterization of “cell wall degrading activity” assigned to phageproteins and capability to convert the degrading activity from a “lytic”function, which is evaluated under artificial conditions, into abactericidal function under non-artificial conditions of typicalbacterial growth circumstances. These “degrading activities” are likelyto be new sources of unrecognized bactericidal activities for use undertherapeutic conditions, and may include muraminidase, glucosaminidase,amidase, or endopeptidase activities. This activity can be identified,isolated, and has been shown, in various exemplary purified solubleprotein constructs, to have bactericidal activity on target bacteria,outside of the context of the phage structures tested under highlyartificial assay conditions. Moreover, recombinant constructs comprisingsuch activities have significant advantageous properties asantimicrobial compositions and formulations.

Similarly, the siphoviridae phage phi11 has a murein-degrading activity(TAME), recognized in part by its pattern of gene structure.

This present study shows that the “hypothesized ORF56” of theStaphylococcus phage K has muralytic activity, and moreover that therecombinant protein product seems to be processed to a 23 kD proteinfrom a 91 kD encoded “putative” translation product. Exposure of variousstrains of Staphylococcus bacteria to the 23 kD product indicatesbactericidal activity. Truncation constructs indicate that thebactericidal activity is encoded in the C-proximal region of the proteintranslation product.

The present studies indicate that a 23 kD protein product which isgenerated from the ORF56 possesses a CHAP domain. Further, a 16 kDtruncated form of the 23 kD protein product encompassing the C-proximalregion and the CHAP domain is also bactericidal. Based upon sequencehomology searches, various other similar structures have been identifiedwhich are potential alternative sources for bactericidal activities.While there may be different muralytic activities, the scope ofbacterial sensitivities are generally unstudied. Thus, various of thesenew activities may have relatively broad target anti-bacterialactivities. Moreover, the small sizes of the polypeptides exhibitingthese activities make them efficient for production and accessibilitywithin a body or to relevant cell wall target components, e.g.,peptidoglycans.

Phage therapy has recently received renewed attention as an alternativefor prevention and/or treatment of bacterial infections. See Merril, etal. (2003) Nat. Rev. Drug Discov. 2:489-497; and Sulakvelidze, et al.(2001) Antimicrob. Agents Chemother. 45:649-659. Alternatively,phage-encoded endolysins have been proposed as effective agents for thecontrol of infectious diseases caused by Gram-positive bacteria(Fischetti (2003) Ann. N.Y. Acad. Sci. 987:207-214). A recent paper onphage endolysins showed non-specificity of Enterococcus phage endolysinswhich act on Streptococcus and Staphylococcus other than Enterococcus(Yoong et al (2004) J. Bact. 186:4808-4812).

These muralytic or cell wall degrading labels assigned to phagecomponents are found in many phage types, including the myoviridae,podoviridae, and siphoviridae classes.

Applicants worked with these TAME cell wall degrading activities testingfor bactericidal activity under the artificial conditions applied to theterm “lysis” entities, and found that their specific activities wererelatively low. It became apparent that this limitation was intrinsic tothe TAME activity, which has a biological role in generating anextremely limited degradation of the cell wall, sufficient to allowinjection of the phage DNA but not to cause a deleterious effect on cellintegrity. In particular, the rate of degradation of the cell wallleading to an effect on the bacterial growth was not striking, and inmost situations was found to be insufficient for commercial therapeuticuse.

Phage proteins have likely evolved to withstand the harsh environmentoutside of cells, and often outside of the body of an animal, and haveevolved for inefficient killing of the target host cell. In fact, thelife cycle of the phage requires that the cell NOT be killed in theinfection process, else the phage life cycle would be aborted beforereplication. Thus, TAME proteins are inherently inefficient asbactericidal elements.

As such, Applicants further recognized that while the phage tail enzymeshave the evolutionary purpose to assist the infection process, they havebeen evolved to NOT be efficient to the extent of killing the targethost. Thus, if TAME proteins were to be useful as efficient bactericidalagents, the TAME proteins would require modification to do so. Thepresent invention provides mutagenesis and screening methods that can beapplied to identify cell wall degrading motifs that can direct TAMEcatalytic domains to a particular bacterium or a particular site on abacterium. Using ORF56 as a model, Applicants were the first torecognize that phage TAME proteins can be converted from a marginalbactericidal agent into a highly efficient and robust bactericidal agentby, e.g., removing sequences which seem to prevent the full-length TAMEpolypeptide from exhibiting high bactericidal function, and/or fusingthe remaining catalytic domain to a heterologous cell-wall bindingdomain. As noted above, these fusions, or chimeras, are here designatedas TAME-CBDs.

Natural forms of these phage TAME proteins have limited bactericidalactivities, as described. Applicants discovered that a targeting motiflinked to the wall degrading domain affected a dramatic increase in thelocal concentration of the catalytic site at the cell wall substrate.Chimeric proteins form specific bacteria can be designed by, e.g.combining a catalytic segment from a TAME protein that acts on theappropriate cell wall structure as found naturally on the bacterialsurface in its natural context and binding segment that has theappropriate affinity and targets the appropriate cell wall structure.Linking a targeting motif to a phage derived cell wall degrading segmentcan provide a number of fusion or bifunctional constructs to screen fordesired bacteriostatic, bactericidal, or cell wall “lytic” activities.

Additional cell wall degrading enzymatic segments have been selected andconstructs made to demonstrate the scope of the present invention. Forexample, segments derived from phage, typically tail structures,encoding enzymatic activities, e.g., cell wall degrading enzymes, havebeen used. Enzymatic activities have been isolated from various phage orbacterial sources, and shown to have similar activities. Similaractivities are available from phage based structures, e.g., based uponsequence homology to known activities used by phage to gain access tothe host, typically in an infection-related process. Others can beidentified by gene organization of infection enzymes, e.g., in cassettescontaining phage tail binding/wall penetration structures, in phagegenomes (see the ORF49 of S. aureus phage phi11 cell wall hydrolase(NP_(—)803302), which is a structural “counterpart” to the ORF56 inphage K. Other examples include ORF004 from S. aureus phage 69(gi:66395297, YP_(—)239591.1), cell wall hydrolase from phage PhiNM4(gi:104641981, ABF73289.1), cell wall hydrolase from phi ETA2(gi:122891778, YP_(—)001004324.1), ORF004 from S. aureus phage 85(gi:66394874, YP_(—)239746.1), and ORF004 from phage ROSA (gi:66395969,YP_(—)240329.1). Both of these domains or motifs may also be derivedfrom prophage or “remnant phage” genomes left in a bacterial genome froman inactivated or incomplete phage genome. Prophages and methods toidentify them are disclosed at, e.g., Canchaya et al., Microbiol. Mole.Biol. Rev. 67:238-276 (2003), which is herein incorporated by referencefor all purposes. Other activities may be derived from pyocins(bacteriocins) or phage related structures which may be incapable ofproliferating as normal phage, but are produced or sustained asbyproducts of incomplete genomes. Thus, proteins or encoding sequencesmay be isolated from structures representing viable phage, or derivedtherefrom. Moreover, each of these structures may serve as a startingpoint for mutagenesis to optimize activities under conditions desiredfor use, e.g., as described.

II. Tail Associated Murein-Degrading Enzymes (TAMES)

Tail Associated Murein-degrading Enzymes (TAMES) are defined asmuralytic enzymes found in the bacteriophage particles and include thosewhich will digest the bacterial cell wall preferably of a Gram-positivebacterium, but may also apply to those which can digest material of aGram-negative or other bacterium. The activity will typically be apeptidoglycan degrading enzyme, and may have one or more muraminidase,glucosaminidase, transglycosylase, lysozyme, amidase or endopeptidaseenzymatic activities. The enzyme may be capable of degrading of the cellwall, and may have even be characterized as “lytic” to the cell, butsuch a lytic characterization is under highly artificial conditions,compared to the normal environment of the phage infection process.Preferably, the enzymes are derived from phage structures, tails ortail-equivalents in podophage, or interior head proteins of podophage,which provide means for the phage genomic material to enter a bacterialhost from the external environment; because these proteins are mostcommonly found in tail structures, for the purposes of this application,the entire class is called the TAME proteins. An example of a TAMEprotein associated with a tail-equivalent in podophage is the gp16protein of Phage T7. The gp16 protein is a transglycosylase that attackspeptidoglycan. The gp16 protein aids in DNA injection, but is containedinside the capsid and when ejected during infection, seems to form partof tail. See, e.g., Molineux (1999) The T7 family of bacteriophages. InEncyclopedia of Molecular Biology. Creighton T E, ed. NY, John Wiley &Col, pp. 2495-2507.

The target bacteria will typically be those which affect or infectanimals, particularly primates. However, various bacteriostatic orbactericidal applications would be advantageously pursued, as willcertain public health problems. The bacteria will often fall into theGram-positive class, though there are other pathological bacteria whichare not clearly categorized into one or the other, includingmycobacteria, spores, or other prokaryotes. Pathogenic or pathologicalbacterial targets are of most interest, both Gram-positive strains,e.g., Staphylococcus species, including aureus, and Streptococcusspecies, as well as Gram-negative. Particularly important Gram-negativetarget species include the genera Escherichia, particularly coli;Pseudomonas, particularly aeruginosa; Campylobacter; Salmonella;Neisseria; Helicobacter; and Vibrio. See, e.g., the Merck Manual and theMerck Veterinary Manual.

The ORF56 polypeptides disclosed herein can be used in combination withat least one other muralytic enzyme to, e.g., treat infection by one ormore bacterial strains. Exemplary additional muralytic enzymes include,e.g., a phage p68 protein 16 and a Pal-type “lytic” enzyme. A phage p68protein 16 is disclosed at, e.g., (Vybiral D et al. (2003), FEMsMicrobiol Lett., 219, 275-283). Pal-type “lytic” enzymes are disclosedat, e.g., Fischetti, et al. (2005) US Pat App 20050208038.

As disclosed herein, TAME proteins can be identified by those of skillthrough a combination of sequence analysis and determination of theposition of the encoding nucleic acid on a phage genome.

III. Definitions

A “cell wall degrading activity” is an enzymatic activity that degrades,breaks down, disintegrates, or diminishes or reduces the integrity of abacterial cell. The term “lytic” is typically used to mean “cell walldegrading”, partly because most (with certain exceptions) of the walldegrading catalytic activities are hydrolytic. Thus, much of theterminology used refers to “lytic” even if the catalytic mechanism doesnot involve hydrolysis. Alternatively degradation of certain defined orartificial substrates may be useful assays for “lytic” or staticactivity (on a populational basis for the target). “Cell wall lyticactivity” in a phage context is usually a characterization assigned to astructure based upon testing under artificial conditions, but suchcharacterization can be specific for bacterial species, families,genera, or subclasses (which may be defined by sensitivity). Therefore,a “bacterium susceptible to a cell wall degrading activity” describes abacterium whose cell wall is degraded, broken down, disintegrated, orthat has its cell wall integrity diminished or reduced by a particularcell wall degrading activity or activities. Many other “lyticactivities” originate from the host bacterial cells, and are importantin cell division or phage release. Other phage derived cell walldegrading activities are found on the phage and have evolved to serve invarious penetration steps of phage infection but would bephysiologically abortive to phage replication if they kill the host cellbefore phage DNA is injected into the cell. The structures useful in thepenetration steps are particularly relevant to the present invention inthat these activities operate on normal hosts from the exterior. Inpreferred embodiments, the cell wall degrading activity is provided byan enzyme that is a non-holin enzyme and/or that is a non-lysin enzyme.In other embodiments, the cell binding activity is provided by an enzymethat is a non-holin enzyme and/or that is a non-lysin enzyme.

A “cell binding domain” or “CBD” is typically a targeting motif, whichrecognizes the bacterial outer surface. In Gram-positive bacteria, theouter surface of the bacteria is typically the murein layer. Thus, thepreferred binding segment for these targets will be cell surfaceentities, whether protein, lipid, sugar, or combination. Bindingsegments from known lysozymes, endolysins, and such are known and theirproperties easily found by PubMed or Entrez searches. Other proteinswhich bind to bacteria include the PGRPs described below, the TLRs,flagellum and pili binding entities, and phage tail proteins involved intarget recognition. In a preferred embodiment, the CBD is fused to aTAME protein or to a cell wall degrading protein, both as disclosedherein. In a further preferred embodiment, the CBD is a heterologousdomain as compared to the TAME protein or to cell wall degradingprotein. That is, the CBD protein is derived from a non-TAME protein ora non-cell wall degrading protein, or is derived from a cell wallbinding protein from a different phage, a bacterium or other organism.Thus, the heterologous CBD domain can be used to direct the TAME proteinto specific target bacteria or can be used to increase the target rangeof the TAME protein.

An “environment” of a bacterium can include an in vitro or an in vivoenvironment. In vitro environments are typically found in a reactionvessel, in some embodiments using isolated or purified bacteria, but caninclude surface sterilization, general treatment of equipment or animalquarters, or public health facilities such as water, septic, or sewerfacilities. Other in vitro conditions may simulate mixed speciepopulations, e.g., which include a number of symbiotically orinteracting species in close proximity. Much of phage and bacterialstudy is performed in cultures in which the ratios of target host andphage are artificial and non-physiological. An in vivo environmentpreferably is in a host organism infected by the bacterium. In vivoenvironments include organs, such as bladder, kidney, lung, skin, heartand blood vessels, stomach, intestine, liver, brain or spinal chord,sensory organs, such as eyes, ears, nose, tongue, pancreas, spleen,thyroid, etc. In vivo environments include tissues, such as gums,nervous tissue, lymph tissue, glandular tissue, blood, sputum, etc., andmay reflect cooperative interactions of different species whose survivalmay depend upon their interactions together. Catheter, implant, andmonitoring or treatment devices which are introduced into the body maybe sources of infection under normal usage. In vivo environments alsoinclude the surface of food, e.g., fish, meat, or plant materials. Meatsinclude, e.g., beef, pork, chicken, turkey, quail, or other poultry.Plant materials include vegetable, fruits, or juices made from fruitsand/or vegetables. In some embodiments surfaces that have come incontact with a bacterially-infected food product are treated with a TAMEprotein or a chimeric protein comprising a TAME protein, e.g., ORF56 orORF49.

“Introducing” a composition to an environment includes administering acompound or composition, and contacting the bacterium with such.Introducing said compound or composition may often be effected by livebacteria which may produce or release such.

A “cell wall degrading protein” is a protein that has detectable, e.g.,substantial, degrading activity on a cell wall or components thereof.“Lytic” activity may be an extreme form or result of the degradingactivity. Exemplary bactericidal polypeptides include, e.g., ORF56 orORF49 products, structurally related entities, mutant and variantsthereof, and other related constructs derived therefrom or from thetwort, K, G1, or phi11 phage. Particular preferred sequences arederived, e.g., from ORF005 from Staph phage G1 (see gi:66394954,YP_(—)240921.1), from ORF007 from Staph phage Twort (see gi:66391262,YP_(—)238566.1), or from Listeria phage P100 (see gi:82547634,YP_(—)406405.1). Similar degrading activities will be identified bytheir location on the phage tails or target host contact points ofnatural phage, mutated phage remnants (e.g., pyocins or bacteriocins),or encoded by prophage sequences. Preferred segments are derived, e.g.,from ORF56 or ORF49, S. simulans lysostaphin (lss), S. aureus LytMpeptidase, S. capitis ALE1, and other phage tail muralytic polypeptides.

An “ORF56 polypeptide” or grammatical variant thereof, refers to abacteriocidal or bacteriocidal activity encoded by the ORF56 ofStaphylococcus phage K (associated structural features are related togi|48696445). Exemplary variant ORF56 polypeptides include polypeptidepolymorphic variants, alleles, mutants, and interspecies homologs that:(1) have an amino acid sequence that has greater than about 60% aminoacid sequence identity, about 65%, 70%, 75%, 80%, 85%, 90%, preferablyabout 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater aminoacid sequence identity, preferably over one or more regions, e.g., of atleast about 8, 12, 17, 25, 33, 50, 65, 80, 100, 200, or more aminoacids, to an amino acid sequence encoded by an ORF56 nucleic acid fromStaphylococcus phage K, see, e.g., Accession Number YP_(—)024486, or toan amino acid sequence of a muralytic polypeptide from Staph phageTwort, K, or 01; (2) bind to antibodies, e.g., polyclonal antibodies,raised against a substantially purified immunogen comprising an aminoacid sequence of an active fragment of ORF56, and conservativelymodified variants thereof; (3) specifically hybridize under stringenthybridization conditions to an anti-sense strand corresponding to anatural nucleic acid sequence encoding the ORF56 polypeptide, andconservatively modified variants thereof; (4) have a nucleic acidsequence that has greater than about 65%, 70%, 75%, 80%, 85%, 90%, or95%, preferably greater than about 96%, 97%, 98%, 99%, or highernucleotide sequence identity, preferably over a region of at least about25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, etc., or morenucleotides, to the ORF56 encoding nucleic acid or a nucleic acidencoding fragment thereof. Particularly preferred segments are derivedfrom the CHAP domain. The nucleic acids and proteins of the inventioninclude both natural or recombinant molecules. The full length ORF56polypeptide and N terminal truncated fragments thereof, as small asabout 16 kD, typically have degradative activity on cell wallcomponents. Assays for degrading activity on cell wall components can beperformed according to methods known to those of skill in the art, andas described herein. In preferred embodiments, ORF56 polypeptide hasbactericidal activity against various Staphylococcus strains ofbacteria, including the aureus, epidermidis, lentis, and carnosusspecies. Analogous measures of comparison may be applicable to othersequences, e.g., ORF49, described herein.

Nucleic acids encoding cell wall degrading polypeptides can, in someembodiments, be amplified using PCR primers based on the sequence ofdescribed cell wall degrading polypeptides. For example, nucleic acidsencoding ORF56 polypeptide variants and fragments thereof, as well aslikely wall degrading activity candidates, can be amplified usingprimers. See, e.g., Vybiral, et al. (2003) FEMS Microbiol. Lett.219:275-283. Thus, cell wall degrading polypeptides and fragmentsthereof include polypeptides that are encoded by nucleic acids that areamplified by PCR based on the sequence of the identified cell walldegrading polypeptides. In a preferred embodiment, a bactericidal orbacteriostatic polypeptide or fragment thereof is encoded by a nucleicacid that is amplified by primers relevant to the ORF56 or ORF49sequences described.

A “phage particle component” refers to, e.g., a head or tail componentof a phage, e.g., Phage K, Twort, 01, or phi11. However, the inventionprovides that many different phage types may be sources of the “lytic”activity loosely ascribed to the phage components. See, e.g., Piuri andHatfull (2006) “A peptidoglycan hydrolase motif within themycobacteriophage TM4 tape measure protein promotes efficient infectionof stationary phase cells” Molecular Microbiology 62:1569-1585. A phagenucleic acid refers to a nucleic acid component of a phage and includesdouble and single stranded nucleic acids, e.g., DNA, RNA, or hybridmolecules. Related sequences may be found in prophages or incompletephage genomes, typically found integrated into the bacterial hostchromosome. Tail components typically mediate the recognition andattachment of the phage to the target host, and may possess cell walldegrading activities which assist in penetration of phage componentsinto the host.

“GMP conditions” refers to good manufacturing practices, e.g., asdefined by the Food and Drug Administration of the United StatesGovernment. Analogous practices and regulations exist in Europe, Japan,and most developed countries.

The term “substantially” in the above definitions of “substantiallypure” generally means at least about 60%, at least about 70%, at leastabout 80%, or more preferably at least about 90%, and still morepreferably at least about 95% pure, whether protein, nucleic acid, orother structural or other class of molecules.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalog refers to a compound that has the same basic chemical structureas a naturally occurring amino acid, e.g., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain a basic chemical structure as anaturally occurring amino acid. Amino acid mimetic refers to a chemicalcompound that has a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid.

“Protein”, “polypeptide”, or “peptide” refers to a polymer in which mostor all of the monomers are amino acids and are joined together throughamide bonds, alternatively referred to as a polypeptide. When the aminoacids are α-amino acids, either the L-optical isomer or the D-opticalisomer can be used. Additionally, unnatural amino acids, e.g.,β-alanine, phenylglycine, and homoarginine, are also included. Aminoacids that are not gene-encoded may also be used in the presentinvention. Furthermore, amino acids that have been modified to includeappropriate structure or reactive groups may also be used in theinvention. The amino acids used in the present invention may be the D-or L-isomer, or mixtures thereof. The L-isomers are generally preferred.In addition, other peptidomimetics are also useful in the presentinvention. For a general review, see, Spatola, in Weinstein, et al.(eds. 1983) CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES ANDPROTEINS, Marcel Dekker, New York, p. 267.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques. In particular, fusions of sequence may be generated,e.g., incorporating an upstream secretion cassette upstream of desiredsequence to generate secreted protein product.

A “fusion protein” refers to a protein comprising amino acid sequencesthat are in addition to, in place of, less than, and/or different fromthe amino acid sequences encoding the original or native full-lengthprotein or subsequences thereof. More than one additional domain can beadded to a cell wall lytic protein as described herein, e.g., an epitopetag or purification tag, or multiple epitope tags or purification tags.Additional domains may be attached, e.g., which may add additional lyticactivities (on the target or associated organisms of a mixed colony orbiofilm), bacterial capsule degrading activities, targeting functions,or which affect physiological processes, e.g., vascular permeability.Alternatively, domains may be associated to result in physical affinitybetween different polypeptides to generate multichain polymer complexes.

The term “nucleic acid” refers to a deoxyribonucleotide, ribonucleotide,or mixed polymer in single- or double-stranded form, and, unlessotherwise limited, encompasses known analogues of natural nucleotidesthat hybridize to nucleic acids in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated or by context, aparticular nucleic acid sequence includes the complementary sequencethereof.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of affecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes typically include at least promoters and/or transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used, e.g., as describedherein. In certain embodiments, an expression cassette can also includenucleotide sequences that encode a signal sequence that directssecretion of an expressed protein from the host cell. Transcriptiontermination signals, enhancers, and other nucleic acid sequences thatinfluence gene expression, can also be included in an expressioncassette. In certain embodiments, a recombinant expression cassetteencoding an amino acid sequence comprising a lytic activity on a cellwall is expressed in a bacterial host cell.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Modification of the heterologous sequence may occur, e.g., bytreating the DNA with a restriction enzyme to generate a DNA fragmentthat is capable of being operably linked to the promoter. Techniquessuch as site-directed mutagenesis are also useful for modifying aheterologous sequence.

The term “isolated” refers to material that is substantially oressentially free from components which interfere with the activity of anenzyme. For a saccharide, protein, or nucleic acid of the invention, theterm “isolated” refers to material that is substantially or essentiallyfree from components which normally accompany the material as found inits native state. Typically, an isolated saccharide, protein, or nucleicacid of the invention is at least about 80% pure, usually at least about90%, and preferably at least about 95% pure as measured by bandintensity on a silver stained gel or other method for determiningpurity. Purity or homogeneity can be indicated by a number of means wellknown in the art. For example, a protein or nucleic acid in a sample canbe resolved by polyacrylamide gel electrophoresis, and then the proteinor nucleic acid can be visualized by staining. For certain purposes highresolution of the protein or nucleic acid may be desirable and, e.g.,HPLC or a similar means for purification may be utilized.

The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence affectstranscription and/or translation of the nucleic acid corresponding tothe second sequence.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or proteins, refers to two or more sequences or subsequences thathave, over the appropriate segment, at least greater than about 60%nucleic acid or amino acid sequence identity, 65%, 70%, 75%, 80%, 85%,90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences thatcorresponds to at least about 13, 15, 17, 23, 27, 31, 35, 40, 50, ormore amino acid residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. Longercorresponding nucleic acid lengths are intended, though codon redundancymay be considered. In a most preferred embodiment, the sequences aresubstantially identical over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these and related algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Current Protocols in Molecular Biology,Ausubel, et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (1995 andSupplements) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul, et al. (1990) J. Mol. Biol.215:403-410 and Altschuel, et al. (1977) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (ncbi.nlm.nih.gov/) or similar sources. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short “words” of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or proteins aresubstantially identical is that the protein encoded by the first nucleicacid is immunologically cross reactive with the protein encoded by thesecond nucleic acid, as described below. Thus, a protein is typicallysubstantially identical to a second protein, for example, where the twopeptides differ only by conservative substitutions. Another indicationthat two nucleic acid sequences are substantially identical is that thetwo molecules hybridize to each other under stringent conditions, asdescribed below.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize to its target subsequence, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. Generally, stringent conditions are selected to beabout 15° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength, pH, and nucleic acid concentration) atwhich 50% of the probes complementary to the target sequence hybridizeto the target sequence at equilibrium. (As the target sequences aregenerally present in excess, at Tm, 50% of the probes are occupied atequilibrium). Typically, stringent conditions will be those in which thesalt concentration is less than about 1.0 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is typically at least twotimes background, preferably 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32-48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90-95° C. for 30-120 sec, an annealing phaselasting 30-120 sec, and an extension phase of about 72° C. for 1-2 min.Protocols and guidelines for low and high stringency amplificationreactions are available, e.g., in Innis, et al. (1990) PCR Protocols: AGuide to Methods and Applications Academic Press, N.Y.

The phrases “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind preferentially to a particular protein and do r′ bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at each position where an arginine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Each polynucleotidesequence described herein which encodes a protein also describespossible silent variations, except where otherwise noted. One of skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and UGG which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule by standard techniques. Accordingly, each “silentvariation” of a nucleic acid which encodes a protein is typicallyimplicit in each described sequence.

Those of skill recognize that many amino acids can be substituted forone another in a protein without affecting the function of the protein,e.g., a conservative substitution can be the basis of a conservativelymodified variant of a protein such as the disclosed cell wall lyticproteins. An incomplete list of conservative amino acid substitutionsfollows. The following eight groups each contain amino acids that arenormally conservative substitutions for one another: 1) Alanine (A),Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V), Alanine (A); 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T), Cysteine(C); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton (1984)Proteins).

Furthermore, one of skill will recognize that individual substitutions,deletions, or additions which alter, add, or delete a single amino acidor a small percentage of amino acids (typically less than 5%, moretypically less than 1%) in an encoded sequence are effectively“conservatively modified variations” where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

One of skill will appreciate that many conservative variations ofproteins, e.g., cell wall lytic proteins, and nucleic acids which encodeproteins yield essentially identical products. For example, due to thedegeneracy of the genetic code, “silent substitutions” (e.g.,substitutions of a nucleic acid sequence which do not result in analteration in an encoded protein) are an implied feature of each nucleicacid sequence which encodes an amino acid. As described herein,sequences are preferably optimized for expression in a particular hostcell used to produce the cell wall lytic proteins (e.g., yeast, human,and the like). Similarly, “conservative amino acid substitutions,” inone or a few amino acids in an amino acid sequence are substituted withdifferent amino acids with highly similar properties, are also readilyidentified as being highly similar to a particular amino acid sequence,or to a particular nucleic acid sequence which encodes an amino acid.Such conservatively substituted variations of any particular sequenceare a feature of the present invention. See also, Creighton (1984)Proteins, W.H. Freeman and Company. In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence generally are also “conservatively modified variations”.

The practice of this invention can involve the construction ofrecombinant nucleic acids and the expression of genes in host cells,preferably bacterial host cells. Optimized codon usage for a specifichost will often be applicable. Molecular cloning techniques to achievethese ends are known in the art. A wide variety of cloning and in vitroamplification methods suitable for the construction of recombinantnucleic acids such as expression vectors are well known to persons ofskill. Examples of these techniques and instructions sufficient todirect persons of skill through many cloning exercises are found inBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);and Current Protocols in Molecular Biology, Ausubel, et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1999 Supplement) (Ausubel). Suitablehost cells for expression of the recombinant polypeptides are known tothose of skill in the art, and include, for example, prokaryotic cells,such as E. coli, and eukaryotic cells including insect, mammalian, andfungal cells (e.g., Aspergillus niger).

Examples of protocols sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR), the ligase chain reaction (LCR), Qβ-replicase amplification andother RNA polymerase mediated techniques are found in Berger, Sambrook,and Ausubel, as well as Mullis, et al. (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis, et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Amheim andLevinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)3:81-94; (Kwoh, et al. (1989) Proc. Nat'l Acad. Sci. USA 86:1173;Guatelli, et al. (1990) Proc. Nat'l Acad. Sci. USA 87:1874; Lomell, etal. (1989) J. Clin. Chem. 35:1826; Landegren, et al. (1988) Science241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace(1989) Gene 4:560; and Barringer, et al. (1990) Gene 89:117. Improvedmethods of cloning in vitro amplified nucleic acids are described inWallace, et al., U.S. Pat. No. 5,426,039.

IV. Commercial Applications

Various applications of the described enzymatic activities can beimmediately recognized. One important application is as antibacterialtreatment of articles which may be contaminated in normal use.Locations, equipment, environments, or the like where target bacteriamay be public health hazards may be treated using such entities.Locations of interest include public health facilities where the purposeor opportunity exists to deal with target bacteria containing materials.These materials may include waste products, e.g., liquid, solid, or air.Aqueous waste treatment plants may incorporate such to eliminate thetarget from effluent, whether by treatment with the enzyme entitiesdirectly, or by release of cells which produce such. Solid waste sitesmay introduce such to minimize possibility of target host outbreaks.Conversely, food preparation areas or equipment need to be regularlycleaned, and the invention provides compositions and means toeffectively eliminate target bacteria. Medical and other publicenvironments subject to contamination may warrant similar means tominimize growth and spread of target microorganisms. The methods may beused in contexts where sterilization elimination of target bacteria isdesired, including air filtration systems for an intensive care unit.

Alternative applications include use in a veterinary or medical context.Means to determine the presence of particular bacteria, or to identifyspecific targets may utilize the effect of selective agents on thepopulation or culture. Inclusion of bacteristatic or bactericidalactivities to cleaning agents, including washing of animals and pets,may be desired.

The ORF56 and related polypeptides can be used to treat bacterialinfections of, e.g., humans or animals. These polypeptides can beadministered prophylactically or can be administered to a subject thathas contracted a bacterial infection. In a preferred embodiment, ORF56polypeptides are used to treat infections caused by bacteria thatreplicate slowly as the killing mechanism does not depend upon host cellreplication. Many antibacterial agents, e.g., antibiotics, are mostuseful against replicating bacteria. Bacteria that replicate slowly havedoubling times of, e.g., about 1-72 hours, 1-48 hours, 1-24 hours, 1-12hours, 1-6 hours, 1-3 hours, or 1-2 hours.

In a preferred embodiment, these proteins are used to treat humans orother animals that are infected with a Staphylococcus species. Inanother preferred embodiment, the ORF56 or ORF49 proteins are used totreat humans or other animals that are infected with amethicillin-resistant Staphylococcus species.

V. Administration

The route of administration and dosage will vary with the infectingbacteria strain(s), the site and extent of infection (e.g., local orsystemic), and the subject being treated. The routes of administrationinclude but are not limited to: oral, aerosol or other device fordelivery to the lungs, nasal spray, intravenous (IV), intramuscular,intraperitoneal, intrathecal, intraocular, vaginal, rectal, topical,lumbar puncture, intrathecal, and direct application to the brain and/ormeninges. Excipients which can be used as a vehicle for the delivery ofthe therapeutic will be apparent to those skilled in the art. Forexample, the enzyme could be in lyophilized form and be dissolved justprior to administration by IV injection. The dosage of administration iscontemplated to be in the range of about 0.03, 0.1, 0.3, 1, 3, 10, 30,100, 300, 1000, 3000, 10000 or more enzyme molecules per bacterium inthe host infection. Depending upon the size of the protein, which mayitself be tandemly associated, or in multiple subunit form (dimer,trimer, tetramer, pentamer, and the like) or in combination with one ormore other entities, e.g., enzymes or fragments of differentspecificity, the dose may be about 1 million to about 10 trillion/perkg/per day, and preferably about 1 trillion/per kg/per day, and may befrom about 10E6 killing units/kg/day to about 10E13 killingunits/kg/day.

Methods to evaluate killing capacity may be similar to methods used bythose of skill to evaluate intact replicating phage, e.g., plaqueforming units or pfu, though killing units may be better evaluated bydetermining the number of surviving bacteria after titration of thekilling units. Killing quantification is more distinct, however, sincenon-replicating phage will not form plaques on bacterial lawns. Thus,serial dilution methods to evaluate the quantity of “killing” units areconveniently used in place of standard pfu. Serial dilutions ofbacterial cultures exposed to the killing compositions can quantifykilling units. Alternatively, comparing total bacterial counts withviable colony units can establish what fraction of bacteria is actuallyviable, and by implication, what fraction have been susceptible to thekilling constructs. Other measures of activity on artificial orspecially prepared substrates can often be used as surrogate measures ofkilling units.

The therapeutic(s) are typically administered until successfulelimination of the pathogenic bacteria is achieved, though broadspectrum formulations may be used while specific diagnosis of theinfecting strain is being determined. Thus the invention contemplatessingle dosage forms, as well as multiple dosage forms of thecompositions of the invention, as well as methods for accomplishingsustained release means for delivery of such single and multi-dosagesforms.

With respect to the aerosol administration to the lungs or other mucosalsurfaces, the therapeutic composition is incorporated into an aerosolformulation specifically designed for administration. Many such aerosolsare known in the art, and the present invention is not limited to anyparticular formulation. An example of such an aerosol is the Proventilinhaler manufactured by Schering-Plough, the propellant of whichcontains trichloromonofluoromethane, dichlorodifluoromethane, and oleicacid. Other embodiments include inhalers that are designed foradministration to nasal and sinus passages of a subject or patient. Theconcentrations of the propellant ingredients and emulsifiers areadjusted if necessary based on the specific composition being used inthe treatment. The number of enzyme killing units to be administered peraerosol treatment will typically be in the range of about 10E6 to 10E13killing units, and preferably about 10E12 killing units.

Methods to evaluate killing capacity are often similar to many methodsused in working with intact replicating phage. In particular, killingquantification is more difficult since the non-replicating phage willnot form plaques on bacteria. Thus, serial dilution methods to evaluatethe quantity of “killing” units will be performed similarly to standardpfu (plaque forming units), but cannot make use of the killing andamplification which occurs on a bacterial lawn. Serial dilutions ofbacterial cultures exposed to the killing compositions can quantifykilling units. Alternatively, comparing total bacterial counts withviable colony units can establish what fraction of bacteria are actuallyviable, and by implication, what fraction have been susceptible to thekilling constructs. Other means for evaluating stasis activity mayinclude release of intracellular contents, whether natural or loaded, orenzymatic activity on defined or prepared substrates which correspond tonatural cell wall structures.

Typically, the killing will decrease bacterial replication capacity byat least about 3 fold, and may affect it by about 10, 30, 100, 300,etc., to many orders of magnitude. However, even slowing the rate ofbacterial replication without killing may have significant therapeuticor commercial value. Preferred genetic inactivation efficiencies may be0.1, 0.2, 0.3, 0.5, 0.8, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 5, 6, 7, 8, ormore log units.

VI. Formulations

The invention further contemplates pharmaceutical compositionscomprising at least one wall degrading enzyme, e.g., muramidase, of theinvention provided in a pharmaceutically acceptable excipient. Theformulations and pharmaceutical compositions of the invention thuscontemplate formulations comprising an isolated enzyme segment specificfor a bacterium; a mixture of two, three, five, ten, or twenty or moreenzymes that affect the same or typical bacterial host; and a mixture oftwo, three, five, ten, or twenty or more enzymes that affect differentbacteria or different strains of the same bacterium, e.g., a cocktailmixture of enzymes that collectively inhibit the growth of multiplestrains of Staphylococcus aureus. In this manner, the compositions ofthe invention can be tailored to the needs of the patient. The compoundsor compositions will typically be sterile or near sterile.

By “therapeutically effective dose” herein is meant a dose that produceseffects, bacteriostatic or preferably bactericidal, for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. See, e.g., Ansel, et al. Pharmaceutical Dosage Formsand Drug Delivery; Lieberman (1992) Pharmaceutical Dosage Forms (vols.1-3), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd(1999) The Art, Science and Technology of Pharmaceutical Compounding;and Pickar (1999) Dosage Calculations. As is known in the art,adjustments for protein degradation, systemic versus localized delivery,and rate of new protease synthesis, as well as the age, body weight,general health, sex, diet, time of administration, drug interaction,spectrum of bacterial components in the colony, and the severity of thecondition may be necessary, and will be ascertainable with someexperimentation by those skilled in the art.

Various pharmaceutically acceptable excipients are well known in theart. As used herein, “pharmaceutically acceptable excipient” includes amaterial which, when combined with an active ingredient of acomposition, allows the ingredient to retain biological activity andwithout causing disruptive reactions with the subject's immune system.Such may include stabilizers, preservatives, salt, or sugar complexes orcrystals, and the like.

Exemplary pharmaceutically carriers include sterile aqueous ofnon-aqueous solutions, suspensions, and emulsions. Examples include, butare not limited to, standard pharmaceutical excipients such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. In other embodiments, thecompositions will be incorporated into solid matrix, including slowrelease particles, glass beads, bandages, inserts on the eye, andtopical forms.

A composition comprising an enzyme of the invention may also belyophilized using means well known in the art, e.g., for subsequentreconstitution and use according to the invention.

Also of interest are formulations for liposomal delivery, andformulations comprising microencapsulated enzymes, including sugarcrystals. Compositions comprising such excipients are formulated by wellknown conventional methods (see, e.g., Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton Pa. 18042,USA).

In general, pharmaceutical compositions can be prepared in variousforms, such as granules, tablets, pills, suppositories, capsules (e.g.adapted for oral delivery), microbeads, microspheres, liposomes,suspensions, salves, lotions and the like. Pharmaceutical grade organicor inorganic carriers and/or diluents suitable for oral and topical usecan be used to make up compositions comprising thetherapeutically-active compounds. Diluents known to the art includeaqueous media, vegetable and animal oils and fats. Formulations mayincorporate stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue.

The pharmaceutical composition can comprise other components in additionto the “lytic” enzyme. In addition, the pharmaceutical compositions maycomprise more than one active ingredient, e.g., two or more, three ormore, five or more, or ten or more different enzymes, where thedifferent enzymes may be specific for the same, different, oraccompanying bacteria. For example, the pharmaceutical composition cancontain multiple (e.g., at least two or more) defined wall degradingenzymes, wherein at least two of the enzymes in the composition havedifferent bacterial specificity. In this manner, the therapeuticcomposition can be adapted for treating a mixed infection of differentbacteria, or may be a composition selected to be effective againstvarious types of infections found commonly in a particular institutionalenvironment. A select combination may result, e.g., by selectingdifferent groups of wall degrading or “lytic” entities derived fromvarious bacteriophage of differing specificity so as to contain at leastone component effective against different or critical bacteria (e.g.,strain, species, etc.) suspected of being present in the infection(e.g., in the infected site). As noted above, the wall degrading enzymecan be administered in conjunction with other agents, such as aconventional antimicrobial agent. In some embodiments, it may bedesirable to administer the enzyme and antibiotic within the sameformulation.

VII. Methodology

Some aspects of practicing the present invention involve well-knownmethods general clinical microbiology, general methods for handlingbacteriophage, and general fundamentals of biotechnology, principles andmethods. References for such methods are listed below and are hereinincorporated by reference for all purposes.

A. General Clinical Microbiology

General microbiology is the study of the microorganisms. See, e.g.,Sonenshein, et al. (eds. 2002) Bacillus Subtilis and Its ClosestRelatives: From Genes to Cells Amer. Soc. Microbiol., ISBN: 1555812058;Alexander and Strete (2001) Microbiology: A Photographic Atlas for theLaboratory Benjamin/Cummings, ISBN: 0805327320; Cann (2001) Principlesof Molecular Virology (Book with CD-ROM; 3d ed.), ISBN: 0121585336;Garrity (ed. 2005) Bergey's Manual of Systematic Bacteriology (2 vol. 2ded.) Plenum, ISBN: 0387950400; Salyers and Whitt (2001) BacterialPathogenesis: A Molecular Approach (2d ed.) Amer. Soc. Microbiol., ISBN:155581171X; Tierno (2001) The Secret Life of Germs: Observations andLessons from a Microbe Hunter Pocket Star, ISBN: 0743421876; Block (ed.2000) Disinfection, Sterilization, and Preservation (5th ed.) LippincottWilliams & Wilkins Publ., ISBN: 0683307401; Cullimore (2000) PracticalAtlas for Bacterial Identification Lewis Pub., ISBN: 1566703921;Madigan, et al. (2000) Brock Biology of Microorganisms (9th ed.)Prentice Hall, ASIN: 0130819220; Maier, et al. (eds. 2000) EnvironmentalMicrobiology Academic Pr., ISBN: 0124975704; Tortora, et al. (2000)Microbiology: An Introduction including Microbiology Place™ Website,Student Tutorial CD-ROM, and Bacteria ID CD-ROM (7th ed.),Benjamin/Cummings, ISBN 0805375546; Demain, et al. (eds. 1999) Manual ofIndustrial Microbiology and Biotechnology (2d ed.) Amer. Soc.Microbiol., ISBN: 1555811280; Flint, et al. (eds. 1999) Principles ofVirology: Molecular Biology, Pathogenesis, and Control Amer. Soc.Microbiol., ISBN: 1555811272; Murray, et al. (ed. 1999) Manual ofClinical Microbiology (7th ed.) Amer. Soc. Microbiol., ISBN: 1555811264;Burlage, et al. (eds. 1998) Techniques in Microbial Ecology Oxford Univ.Pr., ISBN: 0195092236; Forbes, et al. (1998) Bailey & Scott's DiagnosticMicrobiology (10th ed.) Mosby, ASIN: 0815125356; Schaechter, et al. (ed.1998) Mechanisms of Microbial Disease (3d ed.) Lippincott, Williams &Wilkins, ISBN: 0683076051; Tomes (1998) The Gospel of Germs: Men, Women,and the Microbe in American Life Harvard Univ. Pr., ISBN: 0674357078;Snyder and Champness (1997) Molecular Genetics of Bacteria Amer. Soc.Microbiol., ISBN: 1555811027; Karlen (1996) MAN AND MICROBES: Diseaseand Plagues in History and Modern Times Touchstone Books, ISBN:0684822709; and Bergey (ed. 1994) Bergey's Manual of DeterminativeBacteriology (9th ed.) Lippincott, Williams & Wilkins, ISBN: 0683006037.

B. General Methods for Handling Bacteriophage

General methods for handling bacteriophage are well known, see, e.g.,Snustad and Dean (2002) Genetics Experiments with Bacterial VirusesFreeman; O'Brien and Aitken (eds. 2002) Antibody Phage Display: Methodsand Protocols Humana; Ring and Blair (eds. 2000) Genetically EngineeredViruses BIOS Sci. Pub.; Adolf (ed. 1995) Methods in Molecular Genetics:Viral Gene Techniques vol. 6, Elsevier; Adolf (ed. 1995) Methods inMolecular Genetics: Viral Gene Techniques vol. 7, Elsevier; and Hobanand Rott (eds. 1988) Molec. Biol. of Bacterial Virus Systems (CurrentTopics in Microbiology and Immunology No. 136) Springer-Verlag.

C. General Fundamentals of Biotechnology, Principles and Methods

General fundamentals of biotechnology, principles and methods aredescribed, e.g., in Alberts, et al. (2002) Molecular Biology of the Cell(4th ed.) Garland ISBN: 0815332181; Lodish, et al. (1999) Molecular CellBiology (4th ed.) Freeman, ISBN: 071673706X; Janeway, et al. (eds. 2001)Immunobiology (5th ed.) Garland, ISBN: 081533642X; Flint, et al. (eds.1999) Principles of Virology: Molecular Biology, Pathogenesis, andControl, Am. Soc. Microbiol., ISBN: 1555811272; Nelson, et al. (2000)Lehninger Principles of Biochemistry (3d ed.) Worth, ISBN: 1572599316;Freshney (2000) Culture of Animal Cells: A Manual of Basic Technique(4th ed.) Wiley-Liss; ISBN: 0471348899; Arias and Stewart (2002)Molecular Principles of Animal Development, Oxford University Press,ISBN: 0198792840; Griffiths, et al. (2000) An Introduction to GeneticAnalysis (7th ed.) Freeman, ISBN: 071673771X; Kierszenbaum (2001)Histology and Cell Biology, Mosby, ISBN: 0323016391; Weaver (2001)Molecular Biology (2d ed.) McGraw-Hill, ISBN: 0072345179; Barker (1998)At the Bench: A Laboratory Navigator CSH Laboratory, ISBN: 0879695234;Branden and Tooze (1999) Introduction to Protein Structure (2d ed.),Garland Publishing; ISBN: 0815323050; Sambrook and Russell (2001)Molecular Cloning: A Laboratory Manual (3 vol., 3d ed.), CSH Lab. Press,ISBN: 0879695773; and Scopes (1994) Protein Purification: Principles andPractice (3d ed.) Springer Verlag, ISBN: 0387940723.

D. Mutagenesis; Site Specific, Random, Shuffling

Based upon the structural and functional descriptions provide herein,homologs and variants may be isolated or generated which may optimizepreferred features. Thus, additional catalytic segments of phagepenetration functions may be found by structural homology, or byevaluating entities found in characteristic gene organization motifs.Phage tail genes are typically found in particular gene arrangements,and other entities found in the corresponding arrangements can be testedfor a cell wall degrading function. These may also serve as the startingpoints to screen for variants of the structures, e.g., mutagenizing suchstructures and screening for those which have desired characteristics,e.g., broader substrate specificity. Standard methods of mutagenesis maybe used, see, e.g., Johnson-Boaz, et al. (1994) Mol. Microbiol.13:495-504; U.S. Pat. Nos. 6,506,602, 6,518,065, 6,521,453, 6,579,678,and references cited by or therein.

Binding segments may be similarly identified, and prevalent or specifictarget motifs may be screened for binding domains which interactspecifically with them. Many of those targets may be highly expressedproteins, carbohydrate, or lipid containing structures found on thevarious potential target strains. While many proteins are known whichbind to cell walls, two families include the peptidoglycan recognitionproteins (PGRPs, see, e.g., Dziarski and Gupta (2006) “The peptidoglycanrecognition proteins (PGRPs)” Genome Biol. 7:232, PMID: 16930467;Dziarski and Gupta (2006) “Mammalian PGRPs: novel antibacterialproteins” Cell Microbiol. 8:1059-69, PMID: 16819960; Lu, et al. (2006)“Peptidoglycan recognition proteins are a new class of humanbactericidal proteins” J. Biol. Chem. 281:5895-5907; Dziarski (2004)“Peptidoglycan recognition proteins (PGRPs)” Mol. Immunol. 40:877-886,PMID: 14698226; Guan, et al. (2004) “Crystal structure of the C-terminalpeptidoglycan-binding domain of human peptidoglycan recognition proteinIa” J. Biol. Chem. 279:31873-882; Liu, et al. (2001) “PeptidoglycanRecognition Proteins: a novel family of four human innate immunitypattern recognition molecules” J. Biol. Chem. 276:34686-694; and Werner,et al. (2000) “A family of peptidoglycan recognition proteins in thefruit fly Drosophila melanogaster” Proc. Nat'l Acad. Sci. USA97:13772-777) found in species from insects to mammals. There is aconserved segment of about 160 amino acids found at the C-terminus ofthese proteins, and others may be found by PubMed or sequence searches.Another group of proteins which bind to bacteria is the toll-likereceptors (TLR), particularly TLR4 which directly detects bacterial LPS;TLR2 which binds to bacterial lipoproteins, peptidoglycan, and yeastzymosan; TLR3 which binds double stranded RNA; and TLR5, whichrecognizes flagelin, the protein on bacterial flagella. Pili structuresfound on the outside of the bacterial cell may be another structure forwhich proteins target for binding. Mutagenesis may broaden bindingselectivity or increase stability of segments or the entire construct,deletion strategies may eliminate extraneous segments.

The components of the Gram-positive bacteria cell wall may be sharedwith components of the Gram-negative cell wall, or possibly with othermycobacteria or spores. However, there may be additional layers of wallin the Gram-negative which may also serve as additional barriers tophage access. Other activities derived from phage or elsewhere may becombined to penetrate the more complex Gram-negative cell wallstructures. In particular, multiple catalytic segments may providemultiple activities, which may function synergistically, within a singleconstruct, or which can provide synergistic effect when combined withanother therapeutic, e.g., antibiotic or antimicrobial.

A targeting moiety may increase a local concentration of a catalyticfragment, but a linker of appropriate length may also increase thenumber of wall degrading events locally. Thus, linkers compatible withthe target and catalytic motifs or of appropriate length may be usefuland increase the catalytic penetration activity leading to stasis orkilling of target bacteria.

Part of the conceptual advance from the invention is recognition thatphage have been selected to survive outside of cells, often underbiologically inhospitable conditions. Thus, the structures are likely tobe particularly hardy and robust, and resistant to the environmentalconditions which might otherwise inactivate a phage. Bacteria which livein inhospitable environments, e.g., extreme environments of temperature,salt, oxidizing or reactive extremes, high pressure, and others, arelikely to have phage which are particularly adapted to survive outsidethe cells. So these will be hardy, resistant to those extremes, andprobably can survive them more readily than proteins which have not beensubjected to similar selection. And polypeptides derived from thosesources are likely to be more stable in various purification processes,storage, and pharmacological conditions of use. Yet another aspect ofthe invention come from a presumption that the purpose of TAMEstructures is to recognize and bind to the target bacterium, but not tokill the cell quickly. Thus, the TAME have evolved to not be veryefficient at killing under conditions of commercially feasible use. TheORF56 constructs were tested to see whether the marginal commerciallyviable bactericidal activity could be increased. In fact, a combinationof polypeptide deletion and the attachment of a binding functionincreased the activity to a more attractive level of commercialfeasibility. The linkage to a cell wall targeting moiety can increasethe local substrate concentration at the cell wall degrading activesite, and the deletion of sequence from the natural TAME may delete someof the features which may have been adopted to limit the bactericidalrate to prevent killing of the host before the phage can replicatewithin the cell. And these features are found ubiquitously, as arephage, as starting points for collecting and screening for the desiredproperties for these uses.

E. Screening

Screening methods can be devised for evaluating mutants or new candidatefunctional segments. A purified preparation of the phage particles couldbe screened for presence of such gene products on the phage structure.Binding may use crude bacteria cultures, isolated bacterial cell wallcomponents, peptidoglycan preparations, synthetic substrates, orpurified reagents to determine the affinity and number of targetbindings on target cells. Penetration or wall degrading assays may bedevised to evaluate integrity of the cell walls of target strains, lawninhibition assays, viability tests of cultures, activity on cell wallpreparations or other substrates (e.g., as described for bindingmotifs), or release of components (e.g., sugars, amino acids, polymers)of the cell wall upon catalytic action. Amidase activity may be measuredby release of soluble N-acetyl hexose amines (e.g., modifiedMorgan-Elson reaction) or endopeptidase activity by assay for free aminogroups (L-alanine for ala-gly endopeptidases, L-glycine for gly-glyendopeptidases) using a DNFB assay), all three of these assays based onPetit, et al. (1966) “Peptide cross-links in bacterial cell wallpeptidoglycans studied with specific endopeptidases from Streptomycesalbus G” Biochemistry 5:2764-76; PMID: 5968582. Gly-gly endopeptidaseactivity can also be measured as the release of free amino groups fromN-acetylated hexaglycine (acetyl-Gly6), see Kline, et al. (1994) “Acolorimetric microtiter plate assay for lysostaphin using a hexaglycinesubstrate” Anal. Biochem. 217:329-331; PMID: 8203764.

Linker features may be tested to compare the effects on binding orcatalysis of particular linkers, or to compare the various orientationsof fragments. Panels of targets may be screened for catalytic fragmentswhich act on a broader or narrower spectrum of target bacteria, and mayinclude other microbes which may share cell wall components, e.g.,mycobacteria or spores. This may make use of broader panels of relatedStaphylococcus strains, e.g., including carnosus, epidermidis, simulans,and lentis isolates. Strategies may be devised which allow for screeningof larger numbers of candidates or variants.

One method to test for a cell wall degrading activity is to treat thephage with mild detergents or denaturants to release structurallyassociated proteins. These proteins are further tested for walldegrading or “lytic” activity on bacterial cells. Another method is tocheck for cell wall degradation activity or lysis from without (LO) on aphage resistant bacterial host. A third method to assess wall degradingor “lytic” activity associated with phage structural component is toperform Zymogram assays, e.g., where a pure phage preparation iselectrophoresed on SDS-polyacrylamide gel incorporating autoclaved hostbacteria cells. Proteins on the gels are allowed to renature in situ andthen act upon the cell wall components giving rise to clear “lytic”zones when the rest of the gel stains blue with methylene blue dye. See,e.g., Lepeuple, et al, (1998) “Analysis of the bacteriolytic enzymes ofthe autolytic lactococcus lactis subsp. cremoris strain AM2 byrenaturing polyacrylamide gel electrophoresis: identification of aprophage-encoded enzyme” Appl. Environ. Microbiol. 64:4142-428, PMID:9797258. The clear zones are visualized and the protein band from thezones eluted, and identity determined, e.g., by N-terminal sequencing orby Mass spec. ORFs encoding the proteins can then be isolated.

VIII. Isolation of Nucleic Acids Encoding Cell Wall Degradative orBinding Polypeptides

Nucleic acids have been identified that encode the cell wall “lytic” orbinding proteins described above, e.g., Staph phages K, Twort, G1, orphi11, and conservatively modified variants of those sequences. Theencoded cell wall “lytic” proteins have cell wall degrading activity,and those encoding identified CHAP domains are prime candidates,especially those where the CHAP domains are C proximal. Alternativesources include phage tail-like structures (e.g., pyocins or defectivephage-like particles), or genomic sequences which possess characteristicfeatures of “lytic” activity containing elements, e.g., which exhibitthe gene organization characteristic of such structures (see, e.g.,Rybchin (1984) “Genetics of bacteriophage phi 80—a review” Gene 27:3-11;PMID: 6232171).

Examples of nucleic acids that encode cell wall “lytic” polypeptides arealso relevant to the nucleic acid embodiments of the invention. Methodsof obtaining such nucleic acids will be recognized by those of skill inthe art. Suitable nucleic acids (e.g., cDNA, genomic, or subsequences(probes)) can be cloned, or amplified by in vitro methods such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), or the self-sustainedsequence replication system (SSR). Besides synthetic methodologies, awide variety of cloning and in vitro amplification methodologies arewell-known to persons of skill. Examples of these techniques andinstructions sufficient to direct persons of skill through many cloningexercises are found in Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology 152 Academic Press, Inc., San Diego,Calif. (Berger); Sambrook, et al. (1989) Molecular Cloning—A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor Press, NY, (Sambrook, et al.); Current Protocols in MolecularBiology, Ausubel, et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1994 Supplement) (Ausubel); Cashion, et al., U.S. Pat. No. 5,017,478;and Carr, European Patent No. 0,246,864.

A DNA that encodes a cell wall degrading polypeptide, can be prepared bya suitable method described above, including, e.g., cloning andrestriction of appropriate sequences with restriction enzymes. In onepreferred embodiment, nucleic acids encoding cell wall degradingpolypeptides are isolated by routine cloning methods. A nucleotidesequence of a cell wall degrading polypeptide as provided, e.g., inAccession Number YP_(—)024486, can be used to provide probes thatspecifically hybridize to a gene encoding the polypeptide; or to anmRNA, encoding a cell wall degrading protein, in a total RNA sample(e.g., in a Southern or Northern blot). Once the target nucleic acidencoding a cell wall “lytic” protein is identified, it can be isolatedaccording to standard methods known to those of skill in the art (see,e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Vols. 1-3) Cold Spring Harbor Laboratory; Berger and Kimmel (1987)Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques,San Diego: Academic Press, Inc.; or Ausubel, et al. (1987) CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York). Further, the isolated nucleic acids canbe cleaved with restriction enzymes to create nucleic acids encoding thefull-length cell wall degrading polypeptide, or subsequences thereof,e.g., containing subsequences encoding at least a subsequence of acatalytic domain of a cell wall degrading polypeptide. These restrictionenzyme fragments, encoding a cell wall degrading polypeptide orsubsequences thereof, may then be ligated, for example, to produce anucleic acid encoding a cell wall degrading polypeptide.

Similar methods can be used to generate appropriate cell wall bindingfragments or linkers between fragments. Binding segments with affinityto prevalent surface features on target bacteria can be identified andinclude those from, e.g., phage K ORF56, S. simulans lysostaphin. L54aamidase, phage phi11 amidase, S. aureus lysostaphin analogue ALE-1 (seeGI:3287732); bacterial SH3 domain segments found in Staph. aureus NCTC8325 autolysin (see YP_(—)500516), Staph. aureus JH9N-acetylmuramoyl-L-alanine amidase family 2 (see ZP_(—)01242312), Staph.aureus Mu50 amidase (see NP_(—)371437), Staph. aureus RF122phage-related amidase (see YP_(—)417165), Staph. aureus peptidoglycanhydrolase (see AAA26662), Staph. haemolyticus JCSC1435N-acetylmuramoly-L-alanine amidase (see YP_(—)254248), Staph. simulansprotein product CAA29494, bacterial peptidoglycan recognition proteins(PGRPs or PGLYRPs, a large family of highly conserved proteins foundfrom insects to mammals that bind to bacterial peptidoglycan (PGN) ofGram-positive and Gram-negative bacteria), and other related sequences,e.g., homologues by sequence or location in gene cassettes. Bacterialcell walls of various species have been characterized, and proteinswhich bind thereto often are reported, e.g., in PubMed. Often thebinding proteins will possess prokaryotic counterparts of the SarcHomology 3 domains (SH3). Linker segments of appropriate lengths andproperties can be used to connect binding and catalytic domains. See,e.g., Bae, et al. (2005) “Prediction of protein interdomain linkerregions by a hidden Markov model” Bioinformatics 21:2264-2270; andGeorge and Heringa (2003) “An analysis of protein domain linkers: theirclassification and role in protein folding” Protein Engineering15:871-879.

A nucleic acid encoding an appropriate polypeptide, or a subsequencethereof, can be characterized by assaying for the expressed product.Assays based on the detection of the physical, chemical, orimmunological properties of the expressed polypeptide can be used. Forexample, one can identify a cell wall degrading polypeptide by theability of a polypeptide encoded by the nucleic acid to degrade ordigest bacterial cells, e.g., as described herein.

Also, a nucleic acid encoding a desired polypeptide, or a subsequencethereof, can be chemically synthesized. Suitable methods include thephosphotriester method of Narang, et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown, et al. (1979) Meth. Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage, et al. (1981)Tetra. Lett. 22:1859-1862; and the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skillrecognizes that while chemical synthesis of DNA is often limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Nucleic acids encoding a desired polypeptide, or subsequences thereof,can be cloned using DNA amplification methods such as polymerase chainreaction (PCR). Thus, for example, the nucleic acid sequence orsubsequence is PCR amplified, using a sense primer containing onerestriction enzyme site (e.g., NdeI) and an antisense primer containinganother restriction enzyme site (e.g., HindIII). This will produce anucleic acid encoding the desired polypeptide or subsequence and havingterminal restriction enzyme sites. This nucleic acid can then be easilyligated into a vector containing a nucleic acid encoding the secondmolecule and having the appropriate corresponding restriction enzymesites. Suitable PCR primers can be determined by one of skill in the artusing the sequence information provided in GenBank or other sources.Appropriate restriction enzyme sites can also be added to the nucleicacid encoding the cell wall degrading polypeptide or a polypeptidesubsequence thereof by site-directed mutagenesis. The plasmid containinga cell wall degrading polypeptide-encoding nucleotide sequence orsubsequence is cleaved with the appropriate restriction endonuclease andthen ligated into an appropriate vector for amplification and/orexpression according to standard methods. Examples of techniquessufficient to direct persons of skill through in vitro amplificationmethods are found in Berger, Sambrook, and Ausubel, as well as Mullis,et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methodsand Applications (Innis, et al., eds) Academic Press Inc. San Diego,Calif. (1990) (Innis); Arnheim and Levinson (Oct. 1, 1990) C&EN 36-47;The Journal Of NIH Research (1991) 3:81-94; (Kwoh, et al. (1989) Proc.Nat'l Acad. Sci. USA 86:1173; Guatelli, et al. (1990) Proc. Nat'l Acad.Sci. USA 87:1874; Lomell, et al. (1989) J. Clin. Chem. 35:1826;Landegren, et al., (1988) Science 241:1077-1080; Van Brunt (1990)Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; andBarringer, et al. (1990) Gene 89:117.

Some nucleic acids encoding cell wall degrading polypeptides can beamplified using PCR primers based on the sequence of the identifiedpolypeptides.

Other physical properties, e.g., of a recombinant cell wall degradingpolypeptide expressed from a particular nucleic acid, can be compared toproperties of known desired polypeptides to provide another method ofidentifying suitable sequences or domains, e.g., of the cell walldegrading proteins that are determinants of bacterial specificity,binding specificity, and/or catalytic activity. Alternatively, aputative cell wall degrading polypeptide encoding nucleic acid orrecombinant cell wall “lytic” polypeptide gene can be mutated, and itsrole as a cell wall degrading polypeptide, or the role of particularsequences or domains established by detecting a variation in bacterial“lysis” normally enhanced by the unmutated, naturally-occurring, orcontrol cell wall degrading polypeptide. Those of skill will recognizethat mutation or modification of cell wall degrading polypeptides of theinvention can be facilitated by molecular biology techniques tomanipulate the nucleic acids encoding the polypeptides, e.g., PCR. Othermutagenesis or gene shuffling techniques may be applied to thefunctional fragments described herein, including wall degradingactivities, wall binding properties, or linker features compatible withchimeric constructs.

Functional domains of newly identified cell wall degrading polypeptidescan be identified by using standard methods for mutating or modifyingthe polypeptides and testing them for activities such as acceptorsubstrate activity and/or catalytic activity, as described herein. Thesequences of functional domains of the various cell wall degradingproteins can be used to construct nucleic acids encoding or combiningfunctional domains of one or more cell wall degrading polypeptides.These multiple activity polypeptide fusions can then be tested for adesired bactericidal or bacteriostatic activity. Related sequences basedon homology to identified “lytic” activities may be identified andscreened for activity on appropriate substrates. Phage gene organizationfeatures characteristic of the polypeptides found on phage structuresused to attach and penetrate target cell wall structures, e.g., cassettestructures, may identify new sequences which may possess binding and/orbactericidal or bacteriostatic activities useful in attacking the wallfrom outside. Particular examples may include prophage sequences,including incomplete remnants of functional phage genomes, orpyocin-like structures, including particles derived from phage-likegenetic segments, e.g., deletion or mutated genetic remnants of phageremaining in the DNA of a bacterium.

In an exemplary approach to cloning nucleic acids encoding cell walldegrading polypeptides, the known nucleic acid or amino acid sequencesof cloned polypeptides are aligned and compared to determine the amountof sequence identity between them. This information can be used toidentify and select polypeptide domains that confer or modulate cellwall degrading polypeptide activities, e.g., target bacterial or bindingspecificity and/or degrading or “lytic” activity based on the amount ofsequence identity between the polypeptides of interest. For example,domains having sequence identity between the cell wall degradingpolypeptides of interest, and that are associated with a known activity,can be used to construct polypeptides containing that domain and otherdomains, and having the activity associated with that domain (e.g.,bacterial or binding specificity and/or wall degrading activity).Similar strategies may be applied to isolate bacterial SH3 domains whichbind to cell wall structures, peptidoglycan recognizing proteins(PGRPs), phage tail “lytic” polypeptides, or to linkers for spacingbetween domains.

IX. Expression of Desired Polypeptides in Host Cells

Cell wall degrading, or other, proteins of the invention can beexpressed in a variety of host cells, including E. coli, other bacterialhosts, and yeast. The host cells are preferably microorganisms, such as,for example, yeast cells, bacterial cells, or filamentous fungal cells.Examples of suitable host cells include, for example, Azotobacter sp.(e.g., A. vinelandii), Pseudomonas sp., Rhizobium sp., Erwinia sp.,Escherichia sp. (e.g., E. coli), Bacillus, Pseudomonas, Proteus,Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Paracoccus andKlebsiella sp., among many others. The cells can be of any of severalgenera, including Saccharomyces (e.g., S. cerevisiae), Candida (e.g., C.utilis, C. parapsilosis, C. krusei, C. versatilis, C. lipolytica, C.zeylanoides, C. guilliermondii, C. albicans, and C. humicola), Pichia(e.g., P. farinosa and P. ohmeri), Torulopsis (e.g., T. candida, T.sphaerica, T. xylinus, T. famata, and T. versatilis), Debaryomyces(e.g., D. subglobosus, D. cantarellii, D. globosus, D. hansenii, and D.japonicus), Zygosaccharomyces (e.g., Z. rouxii and Z. bailii),Kluyveromyces (e.g., K. marxianus), Hansenula (e.g., H. anomala and H.jadinii), and Brettanomyces (e.g., B. lambicus and B. anomalus).Examples of useful bacteria include, but are not limited to,Escherichia, Enterobacter, Azotobacter, Erwinia, Klebsielia, Bacillus,Pseudomonas, Proteus, and Salmonella.

Once expressed in a host cell, the cell wall degrading polypeptides canbe used to prevent growth of appropriate bacteria. In a preferredembodiment, an ORF56 polypeptide is used to decrease growth of aStaphylococcus bacterium. In a further preferred embodiment, the proteinis used to decrease growth of an S. aureus bacterium, or other similarStaphylococcus species. Fusion constructs combining such fragments maybe generated, including fusion proteins comprising a plurality of walldegrading activities, including both peptidase and amidase catalyticactivities (which may cleave both gly-gly and gly-ala linkages), orcombining the activity with a targeting segment which binds to cell wallstructures. Combinations of degrading activities may act synergisticallyto effect better bacteristatic or bactericidal activity. A linker may beincorporated to provide additional volume for catalytic sites of highlocal concentration near the binding target.

Typically, a polynucleotide that encodes the cell wall degradingpolypeptides is placed under the control of a promoter that isfunctional in the desired host cell. An extremely wide variety ofpromoters is well known, and can be used in expression vectors of theinvention, depending on the particular application. Ordinarily, thepromoter selected depends upon the cell in which the promoter is to beactive. Other expression control sequences such as ribosome bindingsites, transcription termination sites and the like are also optionallyincluded. Constructs that include one or more of these control sequencesare termed “expression cassettes.” Accordingly, the invention providesexpression cassettes into which the nucleic acids that encode fusionproteins, e.g., combining a catalytic fragment with a binding fragment,are incorporated for high level expression in a desired host cell.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change, et al. (1977) Nature 198:1056),the tryptophan (tip) promoter system (Goeddel, et al. (1980) NucleicAcids Res. 8:4057), the tac promoter (DeBoer, et al. (1983) Proc. Nat'lAcad. Sci. USA 80:21-25); and the lambda-derived P_(L) promoter andN-gene ribosome binding site (Shimatake, et al. (1981) Nature 292:128).The particular promoter system is typically not critical to theinvention, many available promoters that function in prokaryotes can beused. A bacteriophage T7 promoter is used in various examples.

For expression of cell wall degrading polypeptides in prokaryotic cellsother than E. coli, a promoter that functions in the particularprokaryotic production species is used. Such promoters can be obtainedfrom genes that have been cloned from the species, or heterologouspromoters can be used. For example, the hybrid trp-lac promoterfunctions in Bacillus in addition to E. coli.

A ribosome binding site (RBS) is conveniently included in the expressioncassettes of the invention. An exemplary RBS in E. coli consists of anucleotide sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon (Shine and Dalgarno (1975) Nature254:34; Steitz in Goldberger (ed. 1979) Biological regulation anddevelopment: Gene expression (vol. 1, p. 349) Plenum Publishing, NY).

For expression of proteins in yeast, convenient promoters includeGAL1-10 (Johnson and Davies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2(Russell, et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J.(1982) 6:675-680), and MFα (Herskowitz and Oshima (1982) in Strathern,et al. (eds.) The Molecular Biology of the Yeast Saccharomyces ColdSpring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209). Anothersuitable promoter for use in yeast is the ADH2/GAPDH hybrid promoter asdescribed in Cousens, et al. (1987) Gene 61:265-275 (1987). Forfilamentous fungi such as, for example, strains of the fungi Aspergillus(McKnight, et al., U.S. Pat. No. 4,935,349), examples of usefulpromoters include those derived from Aspergillus nidulans glycolyticgenes, such as the ADH3 promoter (McKnight, et al. (1985) EMBO J.4:2093-2099) and the tpiA promoter. An example of a suitable terminatoris the ADH3 terminator (McKnight, et al.).

Either constitutive or regulated promoters can be used in the presentinvention. Regulated promoters can be advantageous because the hostcells can be grown to high densities before expression of the fusionproteins is induced. High level expression of heterologous polypeptidesslows cell growth in some situations. An inducible promoter is apromoter that directs expression of a gene where the level of expressionis alterable by environmental or developmental factors such as, forexample, temperature, pH, anaerobic or aerobic conditions, light,transcription factors, and chemicals. Such promoters are referred toherein as “inducible” promoters, which allow one to control the timingof expression of the desired polypeptide. For E. coli and otherbacterial host cells, inducible promoters are known to those of skill inthe art. These include, for example, the lac promoter, the bacteriophagelambda P_(L) promoter, the hybrid trp-lac promoter (Amann, et al. (1983)Gene 25:167; de Boer, et al. (1983) Proc. Nat'l Acad. Sci. USA 80:21),and the bacteriophage T7 promoter (Studier, et al. (1986) J. Mol. Biol.;Tabor, et al. (1985) Proc. Nat'l Acad. Sci. USA 82:1074-78). Thesepromoters and their use are discussed in Sambrook, et al., supra.

A construct that includes a polynucleotide of interest operably linkedto gene expression control signals that, when placed in an appropriatehost cell, drive expression of the polynucleotide is termed an“expression cassette.” Expression cassettes that encode the fusionproteins of the invention are often placed in expression vectors forintroduction into the host cell. The vectors typically include, inaddition to an expression cassette, a nucleic acid sequence that enablesthe vector to replicate independently in one or more selected hostcells. Generally, this sequence is one that enables the vector toreplicate independently of the host chromosomal DNA, and includesorigins of replication or autonomously replicating sequences. Suchsequences are well known for a variety of bacteria. For instance, theorigin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria. Alternatively, the vector can replicate bybecoming integrated into the host cell genomic complement and beingreplicated as the cell undergoes DNA replication.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. A plethora of kits arecommercially available for the purification of plasmids from bacteria(see, e.g., EasyPrepJ, FlexiPrepJ, both from Pharmacia Biotech;StrataCleanJ, from Stratagene; and, QIAexpress Expression System,Qiagen). The isolated and purified plasmids can then be furthermanipulated to produce other plasmids, and used to transfect cells.Cloning in Streptomyces or Bacillus is also possible.

Selectable markers are often incorporated into the expression vectorsused to express the polynucleotides of the invention. These genes canencode a gene product, such as a polypeptide, necessary for the survivalor growth of transformed host cells grown in a selective culture medium.Host cells not transformed with the vector containing the selection genewill not survive in the culture medium. Typical selection genes encodepolypeptides that confer resistance to antibiotics or other toxins, suchas ampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline.Alternatively, selectable markers may encode proteins that complementauxotrophic deficiencies or supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.Often, the vector will have one selectable marker that is functional in,e.g., E. coli, or other cells in which the vector is replicated prior tobeing introduced into the host cell. A number of selectable markers areknown to those of skill in the art and are described for instance inSambrook, et al., supra.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques as described inthe references cited above. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required. To confirm correct sequences in plasmids constructed,the plasmids can be analyzed by standard techniques such as byrestriction endonuclease digestion, and/or sequencing according to knownmethods. Molecular cloning techniques to achieve these ends are known inthe art. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant nucleic acids arewell-known to persons of skill. Examples of these techniques andinstructions sufficient to direct persons of skill through many cloningexercises are found in Berger and Kimmel, Guide to Molecular CloningTechniques Methods in Enzymology, Volume 152, Academic Press, Inc., SanDiego, Calif. (Berger); and Current Protocols in Molecular Biology,Ausubel, et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (1998Supplement) (Ausubel).

A variety of common Vectors suitable for use as starting materials forconstructing the expression vectors of the invention are well known inthe art. For cloning in bacteria, common vectors include pBR322 derivedvectors such as pBLUESCRIPT™, and λ-phage derived vectors. In yeast,vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expression inmammalian cells can be achieved using a variety of commonly availableplasmids, including pSV2, pBC12BI, and p91023, as well as lytic virusvectors (e.g., vaccinia virus, adenovirus, and baculovirus), episomalvirus vectors (e.g., bovine papillomavirus), and retroviral vectors(e.g., murine retroviruses).

The methods for introducing the expression vectors into a chosen hostcell are typically standard, and such methods are known to those ofskill in the art. For example, the expression vectors can be introducedinto prokaryotic cells, including E. coli, by calcium chloridetransformation, and into eukaryotic cells by calcium phosphate treatmentor electroporation. Other transformation methods are also suitable.

Translational coupling may be used to enhance expression. The strategyuses a short upstream open reading frame derived from a highly expressedgene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few aminoacid codons by a termination codon. Just prior to the termination codonis a second ribosome binding site, and following the termination codonis a start codon for the initiation of translation. The system dissolvessecondary structure in the RNA, allowing for the efficient initiation oftranslation. See Squires, et al. (1988) J. Biol. Chem. 263: 16297-16302.

The various polypeptides of the invention can be expressedintracellularly, or can be secreted from the cell. Intracellularexpression often results in high yields. If necessary, the amount ofsoluble, active fusion polypeptide may be increased by performingrefolding procedures (see, e.g., Sambrook, et al., supra; Marston, etal. (1984) Bio/Technology 2:800; Schoner, et al. (1985) Bio/Technology3:151). In embodiments in which the desired polypeptide are secretedfrom the cell, either into the periplasm or into the extracellularmedium, the DNA sequence is often linked to a cleavable signal peptidesequence. The signal sequence directs translocation of the fusionpolypeptide through the cell membrane. An example of a suitable vectorfor use in E. coli that contains a promoter-signal sequence unit ispTA1529, which has the E. coli phoA promoter and signal sequence (see,e.g., Sambrook, et al., supra; Oka, et al. (1985) Proc. Nat'l Acad. Sci.USA 82:7212; Talmadge, et al. (1980) Proc. Nat'l Acad. Sci. USA 77:3988;Takahara, et al. (1985) J. Biol. Chem. 260:2670). In another embodiment,the fusion polypeptides are fused to a subsequence of protein A orbovine serum albumin (BSA), for example, to facilitate purification,secretion, or stability. Affinity methods, e.g., using the target of thebinding fragment may be appropriate.

The cell wall degrading polypeptides of the invention can also befurther linked to other bacterial polypeptide segments, e.g., targetingfragments. This approach often results in high yields, because normalprokaryotic control sequences direct transcription and translation. InE. coli, lacZ fusions are often used to express heterologous proteins.Suitable vectors are readily available, such as the pUR, pEX, and pMR100series (see, e.g., Sambrook, et al., supra). For certain applications,it may be desirable to cleave extraneous sequence from the fusionpolypeptide after purification. This can be accomplished by any ofseveral methods known in the art, including cleavage by cyanogenbromide, a protease, or by Factor X_(a) (see, e.g., Sambrook, et al.,supra; Itakura, et al. (1977) Science 198:1056; Goeddel, et al. (1979)Proc. Nat'l Acad. Sci. USA 76:106; Nagai, et al. (1984) Nature 309:810;Sung, et al. (1986) Proc. Nat'l Acad. Sci. USA 83:561). Cleavage sitescan be engineered into the gene for the fusion polypeptide at thedesired point of cleavage.

More than one recombinant polypeptide may be expressed in a single hostcell by placing multiple transcriptional cassettes in a singleexpression vector, or by utilizing different selectable markers for eachof the expression vectors which are employed in the cloning strategy.

A suitable system for obtaining recombinant proteins from E. coli whichmaintains the integrity of their N-termini has been described by Miller,et al. (1989) Biotechnology 7:698-704. In this system, the gene ofinterest is produced as a C-terminal fusion to the first 76 residues ofthe yeast ubiquitin gene containing a peptidase cleavage site. Cleavageat the junction of the two moieties results in production of a proteinhaving an intact authentic N-terminal reside.

X. Purification of Desired Polypeptides

The polypeptides of the present invention can be expressed asintracellular proteins or as proteins that are secreted from the cell,and can be used in this form, in the methods of the present invention.For example, a crude cellular extract containing the expressedintracellular or secreted polypeptides can be used in the methods of thepresent invention.

Alternatively, the polypeptides can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis and thelike (see, generally, Scopes (1982) Protein PurificationSpringer-Verlag, N.Y.; Deutscher (1990) Methods in Enzymology (vol. 182)Guide to Protein Purification, Academic Press, Inc. NY). Because thedegrading segments, at least, derive from phage proteins selected forstability, purification may make use of these properties to denaturecontaminating materials. Substantially pure compositions of at leastabout 70, 75, 80, 85, 90% homogeneity are preferred, and about 92, 95,98 to 99% or more homogeneity are most preferred. The purifiedpolypeptides may also be used, e.g., as immunogens for antibodyproduction, which antibodies may be used in immunoselection purificationmethods.

To facilitate purification of the polypeptides of the invention, thenucleic acids that encode them can also include a coding sequence for anepitope or “tag” for which an affinity binding reagent is available,e.g., a purification tag. Examples of suitable epitopes include the mycand V-5 reporter genes; expression vectors useful for recombinantproduction of fusion polypeptides having these epitopes are commerciallyavailable (e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-Hisand pcDNA3.1/V5-His are suitable for expression in mammalian cells).Additional expression vectors suitable for attaching a tag to thepolypeptides of the invention, and corresponding detection systems areknown to those of skill in the art, and several are commerciallyavailable (e.g., FLAG, Kodak, Rochester N.Y.). Another example of asuitable tag is a polyhistidine sequence, which is capable of binding tometal chelate affinity ligands. Typically, six adjacent histidines areused, although one can use more or less than six. Suitable metal chelateaffinity ligands that can serve as the binding moiety for apolyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli“Purification of recombinant proteins with metal chelating adsorbents”in Setlow (ed. 1990) Genetic Engineering: Principles and Methods, PlenumPress, NY; commercially available from Qiagen (Santa Clarita, Calif.)).Purification tags also include maltose binding domains and starchbinding domains. Purification of maltose binding domain proteins isknown to those of skill in the art.

Other haptens that are suitable for use as tags are known to those ofskill in the art and are described, for example, in the Handbook ofFluorescent Probes and Research Chemicals (6th ed., Molecular Probes,Inc., Eugene Oreg.). For example, dinitrophenol (DNP), digoxigenin,barbiturates (see, e.g., U.S. Pat. No. 5,414,085), and several types offluorophores are useful as haptens, as are derivatives of thesecompounds. Kits are commercially available for linking haptens and othermoieties to proteins and other molecules. For example, where the haptenincludes a thiol, a heterobifunctional linker such as SMCC can be usedto attach the tag to lysine residues present on the capture reagent.

One of skill would recognize that certain modifications can be made tothe catalytic or functional domains of the polypeptide withoutdiminishing their biological activity. Some modifications may be made tofacilitate the cloning, expression, or incorporation of the catalyticdomain into a fusion polypeptide. Such modifications are well known tothose of skill in the art and include, for example, the addition ofcodons at either terminus of the polynucleotide that encodes thecatalytic domain, e.g., a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionenzyme sites or termination codons or purification sequences.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, e.g., reference to “abacteriophage” includes a plurality of such bacteriophage and referenceto a “host bacterium” includes reference to one or more host bacteriaand equivalents thereof known to those skilled in the art, and so forth.

Publications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.Citations are incorporated herein by reference in their entirety.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Examples I. Full Length ORF56

Accession Number YP_(—)024486 reported a putative ORF56 found in aStaphylococcus phage K. Based upon this report, a full length Phage KORF56 was PCR amplified from an appropriate phage source. Using genespecific primers with an NdeI site in the forward primer and XhoI in thereverse primer, this PCR product was cloned into a pET21a vector under aT7 promoter as an NdeI-XhoI insert. This clone was labeled pGMB617 andcontained the coding region corresponding to amino acids 1 to 808 of theexpected product (SEQ ID NO:1), which should produce a protein productof about 91 kDa.

A. CHAP Domain

The report describing Accession Number YP_(—)024486 identified a domaindescribed as a Cysteine-Histidine dependent Aminohydrolase/Peptidase(CHAP). See, e.g., Rigden, et al. (2003) Trends Biochem. Sci.28:230-234. Certain genes recognized as containing lytic activitiespossess CHAP domains, generally with the domain at the N proximal regionof the encoded polypeptides. The CHAP domain is on the C proximal regionof the putative ORF56 and should correspond to the designated aminoacids from about amino acids 690 to 805.

B. Degradation Product

However, after production and purification, protein products ofapproximately 50 kDa and approximately 23 kDa were present insubstantial amounts as observed by PAGE. These appeared to representstable degradation products of the original 91 kDa expressed protein.

II. Staphylococcus Target Species

Purified protein constructs were initially tested for decrease in CFU(colony forming units) on a Staphylococcus aureus isolate. Certainconstructs were further tested for decrease in CFU on isolates of S.epidermidis, S. lentis, and S. carnosus. It appears that the lyticactivities observed here are less strain specific than many phageinfection selectivities. Thus, it is likely that use of the lyticactivities described herein will also exhibit multiple strainspecificities, and may even be broad across many genera or otherfunctional or structural classes of bacteria, e.g., all Gram-positive oreven including some or all Gram-negative. Moreover, the lytic activitiesmay also be generic to shared structural features between Gram-positiveand Gram-negative classes. For example some features of Gram-negativeinner bacterial cell walls may be shared with the Gram-positive cellwalls.

III. Truncation Constructs

The region described as hypothetical ORF56 has a unique internal PstIsite, whose use could easily generate a construct which would provideapproximately 57 kDa of C terminal region of the protein from aboutamino acids 297 to 808 of SEQ ID NO:1. From the full length ORF56 clonedescribed above, a PstI-HindIII fragment was excised encoding the Cterminal portion of the reading frame. The PstI-HindIII fragment wascloned into a pRSETA vector generating pRSETA-57 kDa (pGMB 599) ORF56clone construct. From this was excised an NdeI-HindIII fragment whichwas cloned into pET21a vector as NdeI-HindIII to generate pGMB 612. Thisclone expressed the 57 kDa protein (expected) as well as about 50 kDaand about 23 kDa proteins. The smaller proteins are, unexpectedly,apparently stable degradation products of about the same size as fromthe construct expressing the full length 91 kDa protein.

A DNA sequence was constructed to produce a 50 kDa C terminal portion ofthe putative ORF56 region corresponding to about amino acids 363 to 808of SEQ ID NO:1. A PCR amplified product was generated using appropriatespecific primers. The PCR product had an NdeI site in the forward primerand an XhoI site in the reverse primer, and the resulting NdeI-XhoIfragment was cloned into pET21a vector to incorporate an NdeI-XhoIinsert. This product was labeled pGDC060/061. This construct expressed aprotein of 50 kDa (as expected) and a protein of about 23 kDa. Again,the smaller protein is, unexpectedly, apparently a stable degraded ORF56protein of about the same size as observed for the constructs of thefull length 91 kDa ORF56 protein and the truncated 57 kDa ORF56 protein.

A DNA construct was generated to produce the 23 kDa C terminal portionof the ORF56 protein corresponding to about Met-(amino acid 603 to 808).DNA sequence of the ORF56 that codes for 23 kDa of the C terminal regionwas PCR amplified introducing an ATG start codon in the forward primer.The PCR product was cloned into pET21a as an NdeI-XhoI fragment togenerate a construct labeled as pGDC070. This construct expressedproteins which run at about 27 kDa on SDS PAGE and another protein whichruns at about 23 kDa. On storage at 4° C., the two forms collapse to asingle band of about 23 kDa.

A DNA construct was generated to produce a 19 kDa C terminal fragment ofthe ORF56 protein corresponding to about amino acids 620 to 808. DNAsequence corresponding thereto was amplified using specific primers andcloned into pET21a as an NdeI-XhoI insert. The resulting construct wasdesignated pGDC089. This construct expressed a single protein that ranon SDS PAGE at about 21 kDa, about the same as the stable degradationproduct observed from the constructs described above.

These various constructs suggest that the 91 kDa full length proteinproduct is not particularly stable under the conditions used. Tworeasonably stable degradation products appear, first a 50 kDa protein,and then a 23 kDa protein. The degradation, whether from a rapidexoprotease activity, from an endoprotease activity, or a combination ofboth is yet unclear. However, it does appear that the differentconstructs are degraded to a stable 23 kDa truncated ORF56 protein.

IV. Antimicrobial Activity of Purified Proteins

The various ORF56 truncations and/or degradation products were testedfor lytic activity using an assay which determined the decrease in CFU(colony forming units) of Staphylococcus aureus bacterial cultures. Inall cases, the ORF56 truncations or degradation products exhibitedsignificant ability to decrease S. aureus CFU in solution, suggestingthat the constructs and stable degradation products all retain lyticactivity on cell walls. The common structural feature in all of theconstructs is the C terminal region, including the CHAP domain.

V. Candidate Homologous Genes with CHAP Domains to be Tested for LyticActivity

ORF56 bactericidal activity correlated with the C-terminal CHAP domain.Therefore, a BLAST search was used to identify additional “lytic”activities in sequenced phage genomes. Other useful sources of these“lytic” segments include components involved in penetration of phagegenome into hosts, e.g., derived from tails or binding components usedby phage to attach to target hosts or from prophage or pyocin-likestructures. Further so called “lytic” activities may be identified asbeing in coding segments for page tail cassettes, e.g., based uponcharacteristic gene organization.

The searches are done using the CHAP domain or other features. Inparticular, those genes where the CHAP domain is at the C-terminalregion of the ORF are more likely to be relevant to this activity. Ofparticular interest are CHAP domain-containing proteins fromStaphylococcal phages K, Twort, and GI.

VI. Chimeric Constructs

A number of fusion constructs were made linking a catalytic fragmentwhich acts on the cell wall of target Staph strains to a targetingfragment which binds to a cell surface entity. The binding moietyprovides selective localization to the surface of the appropriate targetbacterium, and the catalytic activity acts on nearby substrate sites. Alinker may be incorporated, allowing for a broader region of substrateaccessibility (region of high active site concentration). Differentbinding moieties might be used which recognize highly accessible, highlyexpressed, or selective bacterial cell surface markers. Gram-negativecell wall marker binding segments may be found from proteins derivedfrom host bacteria, and similar Gram-negative wall marker bindingsegments may be found from proteins used by them to control cell wallstructure. Phage specific for the hosts should also have tailpolypeptides which recognize and bind their respective host cell wall.Peptidoglycan recognition proteins (PGRPs) from sources ranging from lowto higher eukaryotes and other binding proteins which bind with affinityto particular bacterial cell walls, preferably in physiologicalconditions and form, will be sources for appropriate binding activityfragments. On some circumstances, a plurality of different moietiesmight be employed. Linkers may be selected for ability to allow theother fragments to properly fold without interference while providing atether to increase local catalytic concentration near appropriatesubstrates. Catalytic fragments may target preferred substrates, and aplurality of fragments may target different linkages found on targetbacteria.

In particular, addressing Gram-positive targets, binding segments wouldpreferably originate from proteins which recognize the extracellularcell wall as “exhibited” physiologically by the bacteria. Thus, proteinswhich recognize Gram-negative cell walls may include immune systemcomponents which recognize these infectious agents. An appropriatesource for the cell wall degrading domains will be tail structures fromphage which infect Gram-negative hosts. Likewise, for Gram-positive,binding domains can derive from tail structures from Gram-positiveinfecting phage or from the PGRPs for Gram-negative bacteria. The walldegrading activities may be derived from tail structures that infectGram-negative hosts. To the extent that mycobacteria, spores, or otherprokaryote or related organisms share the structure of the cell wall,these reagents may be useful to modulate their growth.

In addition, because of the selection processes for phage which infectparticular hosts, phage which target hosts which live in extremeconditions, thermophiles, halophiles, conditions of high oxidation orreactive species, pH extremes, highly proteolytic environments, and thelike are particularly interesting sources for useful catalytic orbinding fragments. The proteins which are exposed to the externalenvironment outside the cell (yearning to enter) must have highlyevolved features to survive outside the relatively safe intracellularenvironment. As such, this stability to hostile conditions will selectfor structural features of the domain which will provide great stabilityfor the product. And the product should have good storage properties,may be selected for pharmacological survival and lifetime, and mayprovide simple means for purification and isolation.

Constructs were made comprising various segments from the ORF56 sequence(see GeneID 2948023, YP_(—)024486, YP_(—)024486.1); the 16 KDa fragmentcorresponding to aa669-808; 19 KDa fragment corresponding to aa629-808;13 KDa fragment corresponding to aa691-808; and ORF56 binding fragmentcorresponding to aa629-690. Staphylococcus lysostaphin (lss; AAB53783)segments include the binding fragment corresponding to aa395-493; andcatalytic (lys-lys cleavage) fragment corresponding to aa248-394. AnL54a amidase (AAW38858; YP_(—)185281) binding fragment corresponds toaa376-484. A LytM peptidase (L77194; AAV62278.1) catalytic fragmentcorresponds to aa223-322. A phage phi11 amidase (NP_(—)803306; AAL82281;see 40893-42365 of AF424781.1) fragment corresponds to aa391-490. Theconstructs were driven by a T7 promoter.

A number of fusion constructs were made: Construct 1 has the sequenceMet-(16 KDa ORF56 catalytic fragment)-Leu-Glu-(lysostaphin bindingfragment) and the resulting protein product is referred to as chimera128 (SEQ ID NO:4). Construct 2 has the sequence (19 KDa ORF56 catalyticfragment)-Leu-Glu-(lysostaphin binding fragment). Construct 3 has thesequence (13 KDa ORF56 catalytic fragment)-Leu-Glu-(lysostaphin bindingfragment). Construct 4 has the sequence (16 KDa ORF56 catalyticfragment)-Leu-Glu-(L54a amidase binding fragment). Construct 5 has thesequence Met-(LytM peptidase catalytic fragment)-Leu-Glu-(lysostaphinbinding fragment). Construct 6 has the sequence Met-(lysostaphincatalytic fragment)-(ORF56 binding fragment). Construct 7 has thesequence (LytM peptidase catalytic fragment)-Construct 1, which has twocatalytic domains (LytM peptidase, ORF56). Construct 8 has the sequenceMet-16 KDa ORF56 catalytic fragment-Leu-Glu-(phi11 amidase bindingfragment). Likewise, other catalytic or binding fragments from othersources may be used, or variants of these may be generated and optimizedfor desired features.

The construct 1 was produced in the appropriate host, and the host lysedincluding a sonication step. Similar methods are applied for the otherconstructs. The crude lysate was purified by ammonium sulfateprecipitation (20-50%), Q-500 column chromatography (pH 7.5), CMcellulose chromatography (pH 6.0) using 200 mM NaCl for elution, and gelfiltration. The product was estimated to be >98% pure by silverstaining.

VII. Activity Testing

The construct 1 product, chimera 128, was tested on a panel of 30distinct typed Staphylococcus aureus strains, selected for spa, Agr, orMec types, and including MLST and methicillin resistance. Chimera 128was active on these tested strains, and lawn inhibition was observedwith spotting of 1.5 microgram of protein. Using an MRSA strain B911 atabout 1E8 CFU, full length ORF56 protein at 50 microgram decreased CFUabout 2 log units, while chimera 128 at 1.5 microgram reduced CFU byabout 5 log units (10,000 fold). On various representative strains ofStaph. aureus at 5E5 cells/ml in Mueller Hinton Broth containing 1% BSA(see Kusuma and Kokai-Kun (2005) “Comparison of four methods fordetermining lysostaphin susceptibility of various strains ofStaphylococcus aureus” Antimicrob. Agents Chemother. 49:3256-263; PMID:16048934) incubated at 35° C., colonies were static for 16 hr. Theminimum inhibitory concentration (MIC) for chimera 128 was about 1-10microgram/ml. Testing of survivors of the S. aureus COL strain to afirst exposure with chimera 128 was tested and survivors were found tobe sensitive to protein at reexposure. Testing of alysostaphin-resistant variant of S. aureus strain B911 showed that 99.9%of the cells were susceptible to 1.5 microgram of chimera 128 protein.

The chimera 128 is stable in Tris buffer at 4° C. for at least a month,about 4 weeks at room temperature (about 25° C.), and about 1 day at 37°C. Certain gel and liquid formulations had much longer lengths ofstability.

Additional chimera constructs were tested for activity using lawninhibition assays, zymogram assays, and colony forming unit (CFU) dropassays. A lawn inhibition assay is a qualitative assay where testproteins are spotted onto a lawn of bacteria and growth inhibition zonesare measured. Bactericidal activity corresponds to a zone of inhibitionon the lawn; no activity corresponds to no visible inhibition zone. Azymogram assay is also a qualitative assay where an SDS-PAGE gel isimpregnated with autoclaved target bacterial cells and a phagepreparation is electrophoresed through the gel. Proteins on the gels areallowed to renature in situ and then act upon the cell wall componentsgiving rise to clear “lytic” zones after staining the gel with methyleneblue dye. See, e.g., Lepeuple, et al. (1998) Appl. Environ. Microbiol.64:4142-428, PMID: 9797258. Activity corresponds to visible clear zonesagainst a dark blue background. The CFU drop assay is a quantitativeassay where activity is measured by the percentage killing. Bacterialcultures are mixed with chimera proteins and plated onto LB medium.Activity corresponds to reduction in cell numbers by atleast 99.9%. Noactivity corresponds to no reduction in cell numbers. Appropriatepositive and negative controls are performed with each assay. Resultsfor a number of chimeric proteins are shown in Table 1. Activity wasdemonstrated for a number of TAME-CBD proteins that comprised an ORF56muralytic domain, also referred to as a catalytic domain (CD). ATAME-CBD protein that comprised Lysostaphin CD and an ORF56 bindingdomain also had bactericidal activity.

TABLE 1 Lawn CFU drop CHIMERA inhibition Zymogram assay 16 kDa ORF56-Lysostaphin BD Active Active Active 19 kDa ORF56-LysostaphinBD ActiveActive Active 16 kDa ORF56- Lys17 BD No No No activity activity activity16 kDa ORF56- L54a amidase BD Active Active No activity 13 kDa ORF56-Lysostaphin BD No No No activity activity activity LytM peptidase- 16kDa ORF56- Active Active Active Lysostaphin BD Lysostaphin CD- ORF56 BDActive Active — fusion

Identification of TAME Conserved Domains

We have developed a comprehensive strategy to identify TAME genes inCaudovirales phage genomes. To look for candidate TAME genes, we rely onthe presence in each TAME of a conserved domain (CD) associated withbacterial cell wall binding, a binding domain (CBD) or degradation,muralytic domain (MD). FIG. 1 is an exemplary list of such domains wehave generated from a search of the NCBI CDD (Conserved Domain Database)at its website ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, using thefollowing search keyword string: “lysozyme OR endolysin OR lysin ORmuramidase OR muraminidase OR glucosaminidase OR murein OR peptidoglycanOR cell wall OR lysis OR amidase OR transglycosylase OR autolysin ORhydrolase”. Those of skill will recognize that a variety of searchstrategies using different search terms can be performed. Otherdatabases, can also be searched.

The search product was then manually inspected for relevance tobacterial cell wall binding, maintenance or degradation. A non-limitinglist of Conserved Domains associated with bacterial cell wall bindingfunction (abbreviated CBD for cell binding domain) or degrading function(abbreviated MD for muralytic domain) follows. Any of the conserveddomains listed below can be used in any combination to generate abactericidal chimeric TAME-CBD protein of the invention.

pfam05382: Amidase_(—)5: Bacteriophage peptidoglycan hydrolase. At leastone of the members of this family, the Pal protein from the pneumococcalbacteriophage Dp-1 has been shown to be a N-acetylmuramoyl-L-alanineamidase. According to the known modular structure of this and otherpeptidoglycan hydrolases from the pneumococcal system, the active siteshould reside at the N-terminal domain whereas the C-terminal domainbinds to the choline residues of the cell wall teichoic acids. Thisfamily appears to be related to pfam00877. [pfam05382|68934]. MD

pfam01510: Amidase_(—)2: N-acetylmuramoyl-L-alanine amidase. This familyincludes zinc amidases that have N-acetylmuramoyl-L-alanine amidaseactivity EC:3.5.1.28. This enzyme domain cleaves the amide bond betweenN-acetylmuramoyl and L-amino acids in bacterial cell walls(preferentially: D-lactyl-L-Ala). The structure is known for thebacteriophage T7 structure and shows that two of the conservedhistidines are zinc binding. [pfam01510|65318]. MD

pfam01520: Amidase_(—)3: N-acetylmuramoyl-L-alanine amidase. This enzymedomain cleaves the amide bond between N-acetylmuramoyl and L-amino acidsin bacterial cell walls. [pfam01520|65327]. MD

pfam00912: Transgly: Transglycosylase. The penicillin-binding proteinsare bifunctional proteins consisting of transglycosylase andtranspeptidase in the N- and C-terminus respectively. Thetransglycosylase domain catalyses the polymerization of murein glycanchains. [pfam00912|54762]. MD

cd00737: endolysin_autolysin: Endolysins and autolysins are found inviruses and bacteria, respectively. The ds DNA phages of eubacteria useendolysins or muralytic enzymes in conjunction with hollin, a smallmembrane protein, to degrade the peptidoglycan found in bacterial cellwalls. Similarly, bacteria produce autolysins to facilitate thebiosynthesis of its cell wall hetropolymer peptidoglycan and celldivision. Both endolysin and autolysin enzymes cleave the glycosidicbeta 1,4-bonds between the N-acetylmuramic acid and theN-acetylglucosamine of the peptidoglycan. [cd00737|29561]. MD

pfam07486: Hydrolase_(—)2: Cell Wall Hydrolase. These enzymes have beenimplicated in cell wall hydrolysis, most extensively in Bacillussubtilis. For instance Bacillus subtilis SCLE, the spore cortex-lyticenzyme is expressed during sporulation as an inactive form and thendeposited on the cell outer cortex. During germination the enzyme isactivated and hydrolyses the cortex. A similar role is carried out bythe partially redundant cell wall hydrolase cwlJ. These enzymes may beamidases or peptidases. [pfam07486|70935]. MD

pfam05257: CHAP domain. This domain corresponds to an amidase function.Many of these proteins are involved in cell wall metabolism of bacteria.This domain is found at the N-terminus of a bifunctional Escherichiacoli enzyme, where is functions as a glutathionylspermidine amidaseEC:3.5.1.78. [pfam05257|68816] ORF56 provides an example of a CHAPdomain. MD

pfam03562: MltA: MltA specific insert domain. This beta barrel domain isfound inserted in the MltA a murein degrading transglycosylase enzyme.This domain may be involved in peptidoglycan binding. [pfam03562|67195].MD

pfam01471: PG_binding_(—)1: Putative peptidoglycan binding domain. Thisdomain is composed of three alpha helices. This domain is found at the Nor C terminus of a variety of enzymes involved in bacterial cell walldegradation. This domain may have a general peptidoglycan bindingfunction. This family is found N-terminal to the catalytic domain ofmatrixins. [pfam01471|65280] CBD

pfam08823: PG_binding_(—)2: Putative peptidoglycan binding domain. Thisfamily may be a peptidoglycan binding domain. [pfam08823|72246] CBD

pfam06737: Transglycosylase: Transglycosylase-like domain. This familyof proteins are very likely to act as transglycosylase enzymes relatedto pfam00062 and pfam01464. These other families are weakly matched bythis family, and include the known active site residues.[pfam06737|70216]. MD

pfam06267: DUF1028: Family of unknown function (DUF1028). Family ofbacterial and archaeal proteins with unknown function. Some members areassociated with a C-terminal peptidoglycan binding domain and may beinvolved in peptidoglycan metabolism. [pfam06267|69772]. CBD and MD

pfam01476: LysM: LysM domain. The LysM (lysin motif) domain is about 40residues long. It is found in a variety of enzymes involved in bacterialcell wall degradation. This domain may have a general peptidoglycanbinding function. The structure of this domain is known.[pfam01476|65285]. CBD

smart00701: PGRP: Animal peptidoglycan recognition proteins homologousto Bacteriophage T3 lysozyme. The bacteriophage molecule, but not itsmoth homologue, has been shown to have N-acetylmuramoyl-L-alanineamidase activity. One member of this family, Tag7, is a cytokine.[smart00701|47970]. CBD

COG2951: MltB: Membrane-bound lytic murein transglycosylase B [Cellenvelope biogenesis, outer membrane] [COG2951|32773]. MD

COG2821: MltA: Membrane-bound lytic murein transglycosylase [Cellenvelope biogenesis, outer membrane] [COG2821|32649]. MD

COG0741: MltE: Soluble lytic murein transglycosylase and relatedregulatory proteins (some contain LysM/invasin domains) [Cell envelopebiogenesis, outer membrane] [COG0741|31084]. MD

cd00736: bacteriophage_lambda_lysozyme: The lysozyme from bacteriophagelambda hydrolyses the beta-1,4-glycosidic bond between N-acetylmuramicacid (MurNAc) and N-acetylglucosamine (GlcNAc), as do other lysozymes.But unlike other lysozymes, bacteriophage lambda does not produce areducing end upon cleavage of the peptidoglycan but rather uses the 6-OHof the same MurNAc residue to produce a 1,6-anhydromuramic acid terminalresidue and is therefore a lytic transglycosylase. An identical1,6-anhydro bond is formed in bacterial peptidoglycans by the action ofthe lytic transglycosylases of E. coli. However, they differstructurally. [cd00736|29560]. MD

cd00118: LysM: Lysin domain, found in a variety of enzymes involved inbacterial cell wall degradation. This domain may have a generalpeptidoglycan binding function. [cd00118|29017]. CBD

pfam08230: Cpl-7: Cpl-7 lysozyme C-terminal domain. This domain wasoriginally found in the C-terminal moiety of the Cpl-7 lysozyme encodedby the Streptococcus pneumoniae bacteriophage Cp-7. [pfam08230|71664]CBD and MD

pfam03411: Peptidase_M74: Penicillin-insensitive murein endopeptidase.[pfam03411|67049] 22: pfam01473 CW_binding_(—)1: Putative cell wallbinding repeat. These repeats are characterised by conserved aromaticresidues and glycines are found in multiple. tandem copies in a numberof proteins. The CW repeat is 20 amino acid residues long. These repeatsin Streptococcus phage CP-1 lysozyme may be responsible for the specificrecognition of choline-containing cell walls. Similar but longer repeatsare found in the glucosyltransferases and glucan-binding proteins oforal streptococci and shown to be involved in glucan binding as well asin the related dextransucrases of Leuconostoc mesenteroides. Repeatsalso occur in toxins of Clostridium difficile and other clostridia,though the ligands are not always known. [pfam01473|65282] CBD

pfam01464: SLT: Transglycosylase SLT domain. This family is distantlyrelated to pfam00062. Members are found in phages, type II, type III andtype IV secretion systems (reviewed in). [pfam01464|65274]. MD

pfam00062: Lys: C-type lysozyme/alpha-lactalbumin family.Alpha-lactalbumin is the regulatory subunit of lactose synthase,changing the substrate specificity of galactosyltransferase fromN-acetylglucosamine to glucose. C-type lysozymes are secretedbacteriolytic enzymes that cleave the peptidoglycan of bacterial cellwalls. Structure is a multi-domain, mixed alpha and beta fold,containing four conserved disulfide bonds. [pfam00062|63951]. MD

COG5632: COG5632: N-acetylmuramoyl-L-alanine amidase [Cell envelopebiogenesis, outer membrane] [COG5632|35191 MD

COG5479: COG5479: Uncharacterized protein potentially involved inpeptidoglycan biosynthesis [Cell envelope biogenesis, outer membrane][COG5479|35038]. CBD and MD

COG4623: COG4623: Predicted soluble lytic transglycosylase fused to anABC-type amino acid-binding protein [Cell envelope biogenesis, outermembrane] [COG4623|34243]. CBD and MD

COG3863: COG3863: Uncharacterized distant relative of cellwall-associated hydrolases [COG3863|33653]. CBD and MD

COG3773: SleB: Cell wall hydrolyses involved in spore germination [Cellenvelope biogenesis, outer membrane] [COG3773|33568]. CBD and MD

COG3770: MepA: Murein endopeptidase [Cell envelope biogenesis, outermembrane] [COG3770|33565]. MD

COG3409: COG3409: Putative peptidoglycan-binding domain-containingprotein [Cell envelope biogenesis, outer membrane] [COG3409|33215]. CBD

COG3023: ampD: N-acetyl-anhydromuramyl-L-alanine amidase [Cell envelopebiogenesis, outer membrane] [COG3023|32839]. MD

COG2247: LytB: Putative cell wall-binding domain [Cell envelopebiogenesis, outer membrane] [COG2247|32428]. CBD

COG1215: COG1215: Glycosyltransferases, probably involved in cell wallbiogenesis [Cell envelope biogenesis, outer membrane] [COG1215|31408].CBD

COG0860: AmiC: N-acetylmuramoyl-L-alanine amidase [Cell envelopebiogenesis, outer membrane] [COG0860|31201]. MD

COG0791: Spr: Cell wall-associated hydrolases (invasion-associatedproteins) [Cell envelope biogenesis, outer membrane] [COG0791|31134]. MD

cd02848: Chitinase_N_term: Chitinase N-terminus domain. Chitinaseshydrolyze the abundant natural biopolymer chitin, producing smallerchito-oligosaccharides. Chitin consists of multipleN-acetyl-D-glucosamine (NAG) residues connected via beta-1,4-glycosidiclinkages and is an important structural element of fungal cell wall andarthropod exoskeletons. On the basis of the mode of chitin hydrolysis,chitinases are classified as random, endo-, and exo-chitinases and basedon sequence criteria, chitinases belong to families 18 and 19 ofglycosyl hydrolases. The N-terminus of chitinase may be related to theimmunoglobulin and/or fibronectin type III superfamilies. These domainsare associated with different types of catalytic domains at either theN-terminal or C-terminal end and may be involved inhomodimeric/tetrameric/dodecameric interactions. Members of this familyinclude members of the alpha amylase family, sialidase, galactoseoxidase, cellulase, cellulose, hyaluronate lyase, chitobiase, andchitinase. [cd02848|30335]. MD

cd02847: Chitobiase_C_term: Chitobiase C-terminus domain. Chitobiase(AKA N-acetylglucosaminidase) digests the beta, 1-4 glycosidic bonds ofthe N-acetylglucosamine (NAG) oligomers found in chitin, an importantstructural element of fungal cell wall and arthropod exoskeletons. It isthought to proceed through an acid-base reaction mechanism, in which oneprotein carboxylate acts as catalytic acid, while the nucleophile is thepolar acetamido group of the sugar in a substrate-assisted reaction withretention of the anomeric configuration. The C-terminus of chitobiasemay be related to the immunoglobulin and/or fibronectin type IIIsuperfamilies. These domains are associated with different types ofcatalytic domains at either the N-terminal or C-terminal end and may beinvolved in homodimeric/tetrameric/dodecameric interactions. Members ofthis family include members of the alpha amylase family, sialidase,galactose oxidase, cellulase, cellulose, hyaluronate lyase, chitobiase,and chitinase. [cd02847|30334]. MD

cd00735: bacteriophage_T4-like_lysozyme: Bacteriophage T4-like lysozymeshydrolyse the beta-1,4-glycosidic bond between N-acetylmuramic acid(MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycanheteropolymers of prokaryotic cell walls. Members include a variety ofbacteriophages (T4, RB49, RB69, Aeh1) as well as Dictyostelium.[cd00735|29559]. MD

cd00254: LT_GEWL: Lytic Transglycosylase (LT) and Goose Egg WhiteLysozyme (GEWL) domain. Members include the soluble and insolublemembrane-bound LTs in bacteria, the LTs in bacteriophage lambda, as wellas, the eukaryotic “goose-type” lysozymes (GEWL). LTs catalyze thecleavage of the beta-1,4-glycosidic bond between N-acetylmuramic acid(MurNAc) and N-acetyl-D-glucosamine (GlcNAc), as do “goose-type”lysozymes. However, in addition to this, they also make a new glycosidicbond with the C6 hydroxyl group of the same muramic acid residue.[cd00254|29556]. MD

cd00119: LYZ1: C-type lysozyme (1,4-beta-N-acetylmuramidase, LYZ) andalpha-lactalbumin (lactose synthase B protein, LA). They have a closeevolutionary relationship and similar tertiary structure, however,functionally they are quite different. Lysozymes have primarilybacteriolytic function; hydrolysis of peptidoglycan of prokaryotic cellwalls and transglycosylation. LA is a calcium-binding metalloproteinthat is expressed exclusively in the mammary gland during lactation. LAis the regulatory subunit of the enzyme lactose synthase. Theassociation of LA with the catalytic component of lactose synthase,galactosyltransferase, alters the acceptor substrate specificity of thisglycosyltransferase, facilitating biosynthesis of lactose.[cd00119|29018]. MD

smart00047: LYZ2: Lysozyme subfamily 2; Eubacterial enzymes distantlyrelated to eukaryotic lysozymes. [smart00047|47396]. MD

pfam02016: Peptidase_S66: LD-carboxypeptidase. Muramoyl-tetrapeptidecarboxypeptidase hydrolyses a peptide bond between a di-basic amino acidand the C-terminal D-alanine in the tetrapeptide moiety inpeptidoglycan. This cleaves the bond between an L- and a D-amino acid.The function of this activity is in murein recycling. This family alsoincludes the microcin c7 self-immunity protein. This family correspondsto Merops family S66. [pfam02016|65774]. MD

pfam02324: Glyco_hydro_(—)70: Glycosyl hydrolase family 70. Members ofthis family belong to glycosyl hydrolase family 70 Glucosyltransferasesor sucrose 6-glycosyl transferases (GTF-S) catalyse the transfer ofD-glucopyramnosyl units from sucrose onto acceptor molecules,EC:2.4.1.5. This family roughly corresponds to the N-terminal catalyticdomain of the enzyme. Members of this family also contain the Putativecell wall binding domain pfam01473, which corresponds with theC-terminal glucan-binding domain. [pfam02324|66049]. MD

pfam06347: SH3_(—)4: Bacterial SH3 domain. This family consists ofseveral hypothetical bacterial proteins of unknown function. These arecomposed of SH3-like domains. [pfam06347|69844]. CBD

pfam08239: SH3_(—)3: Bacterial SH3 domain. [pfam08239|71673]. CBD

pfam08460: SH3_(—)5: Bacterial SH3 domain. [pfam08460|71889]. CBD

COG4991: COG4991: Uncharacterized protein with a bacterial SH3 domainhomologue [COG4991|34596]. CBD

COG3103: COG3103: SH3 domain protein [Signal transduction mechanisms][COG3103|32917]. CBD

smart00287: SH3b: Bacterial SH3 domain homologues; [smart00287|47616].CBD

pfam01551: Peptidase_M23: Peptidase family M23. Members of this familyare zinc metallopeptidases with a range of specificities. The peptidasefamily M23 is included in this family, these are Gly-Gly endopeptidases.Peptidase family M23 are also endopeptidases. This family also includessome bacterial lipoproteins for which no proteolytic activity has beendemonstrated. This family also includes leukocyte cell-derivedchemotaxin 2 (LECT2) proteins. LECT2 is a liver-specific protein whichis thought to be linked to hepatocyte growth although the exact functionof this protein is unknown. [pfam01551|65358]. MD

Method of Scanning of Phage Genomes for TAME Candidates

Currently the process is done by manual inspection of each phage genome,although automated scanning may be implemented by CDD [Conserved DomainDatabase in NCBI; or its equivalent] in the future. The step by stepprocess is listed below, using the Staphylococcus phage 11 as anexample.

1. Identify a phage genome in [appropriate database, e.g.,] the GenbankPhage Genomes database (ncbi.nlm.nih.gov/genomes/static/phg.html).Select its reference number (NC number to right of screen; for phage 11,it is NC_(—)004615). [this description is based on using this database,on this date; as the look/feel design evolves, this description thenbecomes “exemplary”]

2. From the Genome Overview window, select the Protein Coding feature[or its functional equivalent] (in this case, 53 proteins). A windowlisting all of the predicted protein products of the genome will open,with the complete predicted protein list. In this case, the geneproducts have been extensively annotated; however, this method does notrequire a previous annotation, other than the automated identificationof potential ORFs.

3. The next step is to examine each predicted protein product for thepresence of one of the CDs [Conserved Domains; i.e., a cell bindingdomain or a muralytic domain] listed above. This manual examinationshould start with the largest predicted protein and proceed down thesize list. In the case of phage 11 [by example], the largest ORF isphi11_(—)45, predicted to encompass 636 aa. The simplest procedure is toselect the 7 digit Gene ID. This brings up an Overview for the ORF,including a graphical display of the ORF showing its location in thephage genome. By selecting this graphical display, a drop-down menu willbe displayed. If there are any CDs in the Orf, one of the choices willbe Conserved Domains. By selecting this option, the ORF will bedisplayed in graphical form with the identity and position of the CDsdetected in its sequence. In the example case of phi11_(—)45, no CD isdetected. This process is repeated for the next largest predictedprotein product; in this case, it would be phi11_(—)44, at 633 aa. Thereare two CDs present in this ORF, but neither belongs to the list shownin FIG. 1. In the example, the next ORF is phi11_(—)49. This ORF turnsout to have two CDs, CHAP and Lyz2, both of which are present in FIG. 1.Ideally, the process should be repeated for all the ORFs greater than150 aa. Generally, a second ORF will yield at least one hit in FIG. 1.In the case of phi11, the ORF phi11_(—)53 is found to have the CHAP,Ami2 and SH3_(—)5 domains.

After the complete list of predicted ORFs has been analyzed, in generaltwo ORFs will be identified: the TAME and the lytic endolysin. Severalcriteria are applied to choose the TAME. First, a TAME will usually bethe largest ORF containing a CD listed in FIG. 1. In the example ofphage 11, the TAME is phi11_(—)49, which is larger than the endolysin,phi11_(—)53 (amidase). A second, confirmatory criterion may be availableif phage tail proteins have been identified. The TAME will be groupedwith the tail genes. In the case of phage 11, the TAME gene,phi11_(—)49, is adjacent to the tail fiber gene, phi11_(—)50, on thesame (+) strand of the DNA, and downstream of other tail genes,including the tape measure gene, phi11_(—)42. The endolysin (in thiscase, amidase; phi11_(—)53), is usually adjacent to or close to theholin (in this case, phi11_(—)52).

TAMEs in Current Staphylococcal Phages

The application of this procedure to the currently availableStaphylococcal phages generated FIG. 1. In this FIGURE, the TAMEcandidate is listed (with its GI number in the far right column) foreach bacteriophage; in the row pertaining to each phage, the CDs used toidentify the TAME are listed.

Once cell binding domains and muralytic domains are identified, those ofskill can, using the disclosure of this specification and standardmolecular biology techniques, generate TAME-CBD proteins. Bactericidalfunction of large numbers of TAME-CBD proteins can be assayed using theassays described herein, e.g., lawn inhibition assays, zymogram assays,and colony forming unit (CFU) drop assays.

Many phage genomes are disclosed in publically available databases. Theidentification of conserved domains from Staphylococcal phages, both CBDand MD, that can be used in chermic TAME-CBD proteins can be extended bythose of skill to identify conserved domains, both CBD and MD, fromphages that infect other bacteria, e.g., phages that infectStreptococcus and Anthrax bacterial strains.

INFORMAL SEQUENCE LISTING SEQ ID NO: 11: YP_024486. Reports hypothetical prot . . . [gi: 48696445]: 1mrrirrpkvr ieivtddntf tlrfedtrdy ngdefgakll gfqtknsmed dssvfqinma 61gdtywdklvm andiirifit pnddpndkeg kqerliqvgm vsqvskvgsy gndqtqfrit 121gqsfvkpfmk fglgviqevq avlpevgwli dgdgdnevkf tgssahevmt giirrfipym 181kynytektyn tidnyldydd lsswdefekl tevsaftnfd gslkqlmdmv tarpfnelff 241knsektpgka qlvlrktpfn ptewraldmi kvptedfiee dvgksdvety siftatpagm 301lkelngdvfs kpqfhpeltd rygytkfeve niylstksgs atedsdssgd dngtergtys 361kimkdlsnyg rdniskgidk ytsklsskyk nlkkaqakki iekfvkegkv tekeyekitg 421nkvddeltsd nrpkltkdkl ksilkekfkt qddfnnskkk kkaktdalke lttkyrfgnk 481thattlldey ikykgeppnd eafdkylkai egvsnvatdt gsdasdsplv mfsrmlfnwy 541hgnpnfyagd iivlgdpkyd lgkrlfiedk qrgdtwefyi esvehkfdyk qgyyttvgvt 601rglkdailed gkgsphrfag lwnqssdfmg glmgedtske lkekgvaekq ssgdkdggsd 661sggaqdggsl dslkkyngkl pkhdpsfvqp gnrhykyqct wyaynrrgql gipvplwgda 721adwiggakga gygvgrtpkq gacviwqrgv qggspqyghv afvekvldgg kkifisehny 781atpngygtrt idmssaigkn aqfiydkk SEQ ID NO: 2of which the ORF seems to run from 58185 to 60611 within the segment:58021 ctggagacat tatcggagga agaattagag aagttctaga tagtaacatg gatatctttg58081 caaatgaaca taagagaagt tattagtaat tttgtattga cacaagagta gtatcatagt58141 atactactct tatacatata aaaaataaaa ggaagtatgt gtat 58185                                                atgcgt agaataagaa 58201gacctaaggt aagaatagaa atagttacag atgataatac atttacattg agatttgaag 58261atacacgaga ctataatggt gatgagtttg gagctaaact tttaggattc caaactaaaa 58321actctatgga agatgatagt tcagttttcc aaataaatat ggcaggagat acttattggg 58381ataagctagt tatggctaat gatatcataa gaatatttat tacacctaat gatgacccta 58441acgataaaga aggaaaacaa gaacgactta tccaggtagg tatggtttct caagtatcaa 58501aagtaggtag ttacggtaat gaccaaactc aatttagaat aacaggtcaa tcttttgtaa 58561aaccttttat gaaatttgga ttaggcgtta ttcaggaagt tcaagctgta ttacctgaag 58621taggttggct tattgatggt gatggagata atgaagtaaa atttactggt agctcagctc 58681atgaagtaat gactggtatt atacgtagat ttatacctta tatgaaatat aactatactg 58741aaaaaacata taatacaatt gataactatc ttgattatga tgatttaagt agttgggatg 58801agtttgaaaa acttacagaa gtttcagcct ttactaattt tgatgggtca ttaaaacagt 58861taatggatat ggtaacagct agacctttta atgagttatt cttcaaaaat tcagaaaaaa 58921cacctggaaa ggctcaactt gtattaagaa agaccccttt taatcctact gagtggagag 58981ctttagatat gattaaagta cctactgagg attttataga agaggatgta ggtaaaagtg 59041atgtagagac atattctata tttacagcaa cacctgcagg tatgttgaaa gagcttaacg 59101gtgatgtatt ttctaaacca caattccacc ctgaattaac tgatagatat ggttatacta 59161aatttgaagt agaaaatatt tatcttagta caaaatcagg ttcagctact gaggattcag 59221attcttcagg tgatgataat ggcacagaac gaggaactta ttctaaaatt atgaaagatt 59281taagtaacta tggaagagat aatatatcta aaggtataga taagtataca agtaaattat 59341cttcaaaata taaaaactta aaaaaagccc aagctaaaaa aattatagag aagtttgtta 59401aagaaggaaa agtaacagaa aaagaatatg aaaaaataac aggtaataag gtagatgatg 59461aattaacatc agataacaga ccgaagttga caaaagataa attaaagagt atactaaaag 59521agaagtttaa aacacaagat gattttaata attctaagaa aaagaaaaaa gctaagacag 59581atgcacttaa agaattgaca actaaatatc gttttggtaa taaaacacat gctacaactt 59641tattagatga atatattaaa tataaaggag agccacctaa cgatgaggct tttgataaat 59701atcttaaagc tattgaaggt gttagtaatg tagctacaga cacaggttca gatgcaagtg 59761atagcccttt agttatgttt tctagaatgc tatttaattg gtatcatggt aaccctaact 59821tctatgcagg agatattatt gttttaggag accctaagta tgacctaggt aaaagattat 59881ttattgaaga taagcaacga ggagacactt gggagttcta tattgaatct gtagaacata 59941aattcgatta taaacagggg tattatacaa ctgtaggagt aactagaggt ttaaaagacg 60001ctattctaga agatggtaaa ggtagtccgc atagatttgc aggattatgg aatcaatcat 60061cagacttcat gggaggtctt atgggtgaag atacttctaa agaacttaaa gaaaaaggtg 60121tagcagagaa acaaagtagt ggagataaag atggtggttc tgatagtggt ggagctcaag 60181atggtggctc tttagattca cttaaaaaat ataacggcaa acttcctaag catgacccaa 60241gttttgttca acctggtaac cgacattata agtatcagtg tacatggtat gcttataata 60301gaagaggtca attaggcata cctgtgcctt tatgggggga cgccgccgac tggataggtg 60361gtgctaaagg agcaggttat ggtgtaggta gaacacctaa acaaggtgct tgtgttatat 60421ggcaaagagg agttcaagga ggtagcccac aatatggtca cgtagcgttt gtagagaaag 60481tattagatgg aggtaaaaaa atatttatct ctgaacataa ctatgctacc cctaatggat 60541atggtactag aacgatagat atgagttcag ccataggtaa gaatgcacaa ttcatttacg 60601ataagaaata a 60612            aggaggata gtctatggca acagataaag aagctaaaga tgttattgat 60661aaatttatag acaatgtatt taattttgat gtacttacaa aagaaagaat aaaagaaaaa 60721gatgaagaaa ttaaaaaaat aactacagat gatatgtatg aaaaggttgt gtatatacga 60781ccttatgttg gagtaataca aagccttaac cctcagcatg ttcagtatga atcattttct 60841aataatggtt atgatataga ggcagaatta agtttcagga aagtaagtta tttagttgat 60901aaagggtcta tacctacaga ttctttatct actttaacag ttcatttagt agaacgaaat 60961caagaactat taatagatta ctttgatgag atacaagatg tgttgtatgg agaatatatg 61021gaagaagaat atgtatttga tgaagatgta ccattaagta cgatactagc attagacttaSEQ ID NO: 3 NP_803302 (ORF49 of phage phi11) 1mglpnpknrk ptasevvewa lyiaknkiai dvpgsgmgaq cwdlpnylld kywgfrtwgn 61adamaqksny rgrdfkiirn tkdfvpqpgd wgvwtggwag hvnivvgpct kdywygvdqn 121wytnnatgsp pykikhsyhd gpgggvkyfv rppyhpdktt papkpeddsd dneknnkkvp 181iwkdvttiky tissqevnyp eyiyhfiveg nrrlekpkgi mirnaqtmss veslynsrkk 241ykqdveyphf yvdrhniwap rravfevpne pdyividvce dysasknefi fneihamvva 301vdmmakyeip lsienikvdd siwrsmlehv nwnmidngvp pkdkyealek allnifknre 361kllnsitkpt vtksrikvmv dnknadianv rdssptanng saskqpqiit etspytfkqa 421ldkqmargnp kksnawgwan atraqtssam nvkriwesnt qcyqmlnigk yqgvsvsaln 481kilkgkgtln nqgkafaeac kkhnineiyl iahaflesgy gtsnfangkd gvynyfgiga 541ydnnpnyamt farnkgwtsp akaimggasf vrkdyinkgq ntlyrirwnp knpathqyat 601aiewcqhqas tiaklykqig lkgiyftrdk yk SEQ ID NO: 4 Chimera 128M S L D S L K K Y N G K L P K H D P S F V Q P G N R H Y K Y Q C T W Y A YN R R G Q L G I P V P L W G D A A D W I G G A K G A G Y G V G R T P K Q GA C V I W Q R G V Q G G S P Q Y G H V A F V E K V L D G G K K I F I S E H NY A T P N G Y G T R T I D M S S A I G K N A Q F I Y D K K L E T P N T G W KT N K Y G T L Y K S E S A S F T P N T D I I T R T T G P F R S M P Q S G V L KA G Q T I H Y D E V M K Q D G H V W V G Y T G N S G Q R I Y L P V R T W NK S T N T L G V L W G T I K SEQ ID NO: 5Lysostaphin BD fused to the C-ter of 16 kDa ORF56M S L D S L K K Y N G K L P K H D P S F V Q P G N R H Y K Y Q C T W Y A Y N R RG Q L G I P V P L W G D A A D W I G G A K G A G Y G V G R T P K Q G A C V I W QR G V Q G G S P Q Y G H V A F V E K V L D G G K K I F I S E H N Y A T P N G Y G TR T I D M S S A I G K N A Q F I Y D K K L E T P N T G W K T N K Y G T L Y K S E SA S F T P N T D I I T R T T G P F R S M P Q S G V L K A G Q T I H Y D E V M K Q D GH V W V G Y T G N S G Q R I Y L P V R T W N K S T N T L G V L W G T I KSEQ ID NO: 6 Lysostaphin BD fused to the C-ter of 19 kDa ORF56M G G L M M G E D T S K E L K E K G V A E K Q S S G D K D G G S D S G G A Q D GG S L D S L K K Y N G K L P K H D P S F V Q P G N R H Y K Y Q C T W Y A Y N R RG Q L G I P V P L W G D A A D W I G G A K G A G Y G V G R T P K Q G A C V I W QR G V Q G G S P Q Y G H V A F V E K V L D G G K K I F I S E H N Y A T P N G Y G TR T I D M S S A I G K N A Q F I Y D K K L E T P N T G W K T N K Y G T L Y K S E SA S F T P N T D I I T R T T G P F R S M P Q S G V L K A G Q T I H Y D E V M K Q D GH V W V G Y T G N S G Q R I Y L P V R T W N K S T N T L G V L W G T I KSEQ ID NO: 7Lysostaphin BD fused to the C-ter of 131 cDa CHAP domain ORF56G N R H Y K Y Q C T W Y A Y N R R G Q L G I P V P L W G D A A D W I G G A K GA G Y G V G R T P K Q G A C V I W Q R G V Q G G S P Q Y G H V A F V E K V L D GG K K I F I S E H N Y A T P N G Y G T R T I D M S S A I G K N A Q F I Y D K K L E TP N T G W K T N K Y G T L Y K S E S A S F T P N T D I I T R T T G P F R S M P Q S GV L K A G Q T I H Y D E V M K Q D G H V W V G Y T G N S G Q R I Y L P V R T W NK S T N T L G V L W G T I K SEQ ID NO: 8Phage L54 a amidase BD fused to the C-ter of 16 kDa ORF56M A Q D G G S L D S L K K Y N G K L P K H D P S F V Q P G N R H Y K Y Q C T W YA Y N R R G Q L G I P V P L W G D A A D W I G G A K G A G Y G V G R T P K Q G AC V I W Q R G V Q G G S P Q Y G H V A F V E K V L D G G K K I F I S E H N Y A T PN G Y G T R T I D M S S A I G K N A Q F I Y D K K L E K T S A K N Q K N P P V P AG Y T L D K N N V P Y K K E Q G N Y T V A N V K G N N V R D G Y S T N S R I T GV L P N N T T I T Y D G A Y C I N G Y R W I T Y I A N S G Q R R Y I A T G E V D K AG N R I S S F G K F S T I SEQ ID NO: 9LytM peptidase domain fused to the lysostaphin BD at C-terMPENSPVYSLTDGTVVQAGWSNYGGGNQVTEKEANSNNYQWYMHNNRLTVSAGDKVKAGDQIAYSGSTGNSTAPHVHFQRMSGGIGNQYAVDPTSYLQSR L E T P N T GW K T N K Y G T L Y K S E S A S F T P N T D I I T R T T G P F R S M P Q S G V L K AG Q T I H Y D E V M K Q D G H V W V G Y T G N S G Q R I Y L P V R T W N K S T NT L G V L W G T I K SEQ ID NO: 10The catalytic domain of lysostaphin fused to the binding domain of ORF56M A A T H E H S A Q W L N N Y K K G Y G Y G P Y P L G I N G G M H Y G V D F FM N I G T P V K A I S S G K I V E A G W S N Y G G G N Q I G L I E N D G V H R Q WY M H L S K Y N V K V G D Y V K A G Q I I G W S G S T G Y S T A P H L H F Q R MV N S F S N S T A Q D P M P F L K S A G Y G K A G G T V M G G L M M G E D T S KE L K E K G V A E K Q S S G D K D G G S D S G G A Q D G G S L D S L K K Y N G KL P K H D P S F V Q P SEQ ID NO: 11LytM peptidase- 16 kDa ORF56- Lysostaphin BD fusionMPENSPVYSLTDGTVVQAGWSNYGGGNQVTIKEANSNNYQWYMHNNRLTVSAGDKVKAGDQIAYSGSTGNSTAPHVHFQRMSGGIGNQYAVDPTSYLQSRM S L D S L KK Y N G K L P K H D P S F V Q P G N R H Y K Y Q C T W Y A Y N R R G Q L G I P VP L W G D A A D W I G G A K G A G Y G V G R T P K Q G A C V I W Q R G V Q G GS P Q Y G H V A F V E K V L D G G K K I F I S E H N Y A T P N G Y G T R T I D M S SA I G K N A Q F I Y D K K L E T P N T G W K T N K Y G T L Y K S E S A S F T P N TD I I T R T T G P F R S M P Q S G V L K A G Q T I H Y D E V M K Q D G H V W V G YT G N S G Q R I Y L P V R T W N K S T N T L G V L W G T I KSEQ ID NO: 12 16 kDa ORF56- phi11 amidase BDM S L D S L K K Y N G K L P K H D P S F V Q P G N R H Y K Y Q C T W Y A Y N R RG Q L G I P V P L W G D A A D W I G G A K G A G Y G V G R T P K Q G A C V I W QR G V Q G G S P Q Y G H V A F V E K V L D G G K K I F I S E H N Y A T P N G Y GTR T I D M S S A I G K N A Q F I Y D K K L EPVASAWKRNKYGTYYMEESARFTNGNQPITVRKVGPFLSCPVGYQFQPGGYCDYTEVMLQDGHVWVGYTWEGQRYYLPIRTWNGSAPPNQILGDLWGEIS

1. A chimeric Tail Associated Muralytic Enzyme (TAME) polypeptide,wherein the chimeric TAME polypeptide comprises a muralytic domain (MD)and a heterologous cell binding domain (CBD) that binds to a targetbacterium, wherein the target bacterium exhibits reduced or no growthafter being contacted with the chimeric TAME polypeptide.
 2. Thechimeric TAME polypeptide of claim 1, wherein the MD is a catalyticdomain from an ORF49 protein.
 3. The chimeric TAME polypeptide of claim1, wherein the MD is a catalytic domain from an ORF56 protein.
 4. Thechimeric TAME polypeptide of claim 1, wherein the MD is selected from acatalytic domain from a lysostaphin protein, a catalytic domain from anORF56 protein, a catalytic domain from an ORF49 protein, and a catalyticdomain from a LytM peptidase.
 5. The chimeric TAME polypeptide of claim1, wherein the target bacterium is a Staphylococcus species.
 6. Thechimeric TAME polypeptide of claim 5, wherein the target bacterium is amethicillin-resistant Staphylococcus species.
 7. The chimeric TAMEpolypeptide of claim 1, wherein the target bacterium is a slowlyreplicating bacterial species.
 8. A method of enzymatically degrading acell wall of a bacterium, the method comprising contacting the cell wallwith the chimeric TAME polypeptide of claim
 1. 9. A method of treating abacterial infection in a subject in need of such treatment, the methodcomprising administering a pharmaceutical composition comprising thechimeric TAME polypeptide of claim 1 to the subject. 10-14. (canceled)15. The chimeric TAME polypeptide of claim 1, wherein the CBD is from aprotein that is a member selected from the group consisting of an ORF56protein and a lysostaphin protein.
 16. The chimeric TAME polypeptide ofclaim 1, wherein the MD is a catalytic domain from an ORF56 protein andthe CBD is from a lysostaphin protein.
 17. A method of disinfecting asurface, the method comprising the step of contacting the surface withthe chimeric TAME polypeptide of claim
 1. 18. A substantially pure orisolated polypeptide: a) comprising at least 85% identity over a segmentof at least 17 amino acids to residues 1-808, 297-808, 363-808, 603-808,620-808, or 691-805 of ORF56; b) comprising at least 90% identity over asegment of at least 24 amino acids to residues 691-805 of ORF56; or c)comprising a plurality of distinct segments of a least 85% identity toORF56, which segments do not overlap. 19-27. (canceled)
 28. An isolatedbacteriocidal polypeptide comprising a) a Tail AssociatedMurein-degrading Enzyme (TAME) polypeptide, wherein the TAME polypeptidedegrades the cell wall of a bacterium, and b) heterologous targetingdomain, wherein the targeting domain binds to the bacterial cell wall.29. A nucleic acid that encodes the isolated bacteriocidal polypeptideof claim
 28. 30. A host cell comprising the nucleic acid of claim 29.31. A method of killing a bacterium, the method comprising the step ofcontacting the bacterium with the isolated bacteriocidal polypeptide ofclaim
 28. 32. The isolated bacteriocidal polypeptide of claim 28,wherein the TAME polypeptide degrades the cell wall of a Gram-positivebacterium and the targeting domain binds to the cell wall of theGram-positive bacterium. 33-44. (canceled)